U.S. patent application number 12/818017 was filed with the patent office on 2011-01-27 for crystallization of anti-cd20 antibodies.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Timothy Breece, Brian Lobo, Shadia Abike Oshodi, James A. Wilkins.
Application Number | 20110020322 12/818017 |
Document ID | / |
Family ID | 40347971 |
Filed Date | 2011-01-27 |
United States Patent
Application |
20110020322 |
Kind Code |
A1 |
Wilkins; James A. ; et
al. |
January 27, 2011 |
CRYSTALLIZATION OF ANTI-CD20 ANTIBODIES
Abstract
The present invention relates generally to crystalline forms of
anti-CD20 antibodies and purification of anti-CD20 antibodies
involving crystallization.
Inventors: |
Wilkins; James A.; (San
Francisco, CA) ; Oshodi; Shadia Abike; (New York,
NY) ; Lobo; Brian; (Carlsbad, CA) ; Breece;
Timothy; (San Francisco, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
40347971 |
Appl. No.: |
12/818017 |
Filed: |
June 17, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2008/087008 |
Dec 16, 2008 |
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12818017 |
|
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61016288 |
Dec 21, 2007 |
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Current U.S.
Class: |
424/130.1 ;
530/387.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/065 20130101; C07K 2299/00 20130101; C07K 16/2887
20130101 |
Class at
Publication: |
424/130.1 ;
530/387.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00 |
Claims
1. A method of purifying a CD20 antibody from a mixture, comprising
crystallizing the CD20 antibody and recovering the CD20 antibody
from said mixture.
2. The method of claim 1 wherein said mixture has not been
subjected to prior lyophilization.
3. The method of claim 1 wherein said mixture is concentrated
Harvested Cell Culture Fluid (HCCF) of a recombinant cell culture
producing the CD20 antibody.
4. The method of claim 3 wherein the cell culture is a mammalian
cell culture.
5. The method of claim 4 wherein the mammalian cells are Chinese
Hamster Overy (CHO) cells.
6. The method of claim 4 wherein the purification is performed in
the absence of a Protein A purification step.
7. The method of claim 6 wherein the purification is performed in
the absence of a cation exchange chromatography step.
8. The method of claim 4 wherein the purification additionally
comprises a viral filtration step and an anion exchange
chromatography step.
9. The method of claim 8 comprising the steps of (a) crystallizing
the CD20 antibody, (b) dissolving the CD20 antibody crystals in a
buffer, (c) subjecting the solution obtained from step (b) to anion
exchange chromatography, and (d) concentrating the eluate obtained
from the anion exchange chromatography.
10. A method of purifying a CD20 antibody from concentrated
Harvested Cell Culture Fluid (HCCF) of mammalian cells, comprising
the steps of (a) concentrating the HCCF, (b) crystallizing the CD20
antibody, (c) dissolving the CD20 crystals to obtain a CD20
solution, (d) subjecting the CD20 solution to purification on an
anion exchange column, and (c) isolating the CD20 antibody.
11. The method of claim 10 wherein the CD20 antibody is a 2H7
antibody.
12. The method of claim 11 wherein the CD20 antibody is selected
from the group consisting of 2H7 CD20 antibody variants A-I listed
in Table 1.
13. The method of claim 12 wherein the CD20 antibody is selected
from the group consisting of 2H7 CD20 antibody variants A, C and H
listed in Table 1, having VL and VI-1 pairs of SEQ ID NOs: 1 and 2;
SEQ ID NOs: 3 and 4; and SEQ ID NOs: 3 and 5, respectively.
14. The method of claim 11 wherein the HCCF is concentrated to a
CD20 antibody concentration at about or greater than 1.5 mg/ml.
15. The method of claim 10 wherein crystallization is performed at
a pH of about 6.0 to about 8.0.
16. The method of claim 15 wherein crystallization is performed at
a pH of 7.8+/-0.2.
17. The method of claim 10 wherein crystallization is performed at
a temperature of about 4.degree. C. to about 40.degree. C.
18. The method of claim 17 wherein crystallization is performed at
a temperature of about 37.degree. C.
19. The method of claim 10 wherein crystallization is induced by
one or more precipitant selected from the group consisting of PBS,
NaCl, Na.sub.2SO.sub.4, KCl, K.sub.2SO.sub.4, Na.sub.2HPO.sub.4,
and KH.sub.2PO.sub.4.
20. The method of claim 19 wherein the precipitant is
KH.sub.2PO.sub.4.
21. A method of purifying a CD20 antibody from concentrated
Harvested Cell Culture Fluid (HCCF) of mammalian cells, comprising
the steps of (a) concentrating the HCCF, (b) diafiltering the HCCF
with a high salt concentration at a pH that inhibits
crystallization, (c) crystallizing the CD20 antibody by raising the
pH, (d) dissolving the CD20 antibody crystals to obtain a CD20
antibody solution, (e) subjecting the CD20 antibody solution to
purification on an anion exchange column, and (f) isolating the
resultant purified CD20 antibody.
22. The method of claim 21 wherein the CD20 antibody is selected
from the group consisting of 2H7 CD20 antibody variants A-I listed
in Table 1.
23. The method of claim 22 wherein the CD20 antibody is selected
from the group consisting of 2H7 CD20 antibody variants A, C and H
listed in Table 1, having VL and VH pairs of SEQ ID NOs: 1 and 2;
SEQ ID NOs: 3 and 4; and SEQ ID NOs: 3 and 5, respectively.
24. A crystal of a CD20 antibody.
25. The crystal of claim 24 which has a microneedle, needle,
globular or globular peanut morphology.
26. A composition comprising a crystal of claim 24.
27. The composition of claim 26 which is a pharmaceutical
composition, comprising one or more pharmaceutically acceptable
excipients.
28. A method for treating a CD20-associated condition or disease
comprising administering to a mammalian subject an effective amount
of a CD20 antibody purified by the method of claim 1 or claim 21.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to crystalline forms
of anti-CD20 antibodies and purification of anti-CD20 antibodies
involving crystallization.
BACKGROUND OF THE INVENTION
CD20 Antibodies
[0002] Rituximab (RITUXAN.RTM.) is a genetically engineered
chimeric murine/human monoclonal antibody directed against the CD20
antigen. Rituximab is the antibody called "C2B8" in U.S. Pat. No.
5,736,137 issued Apr. 7, 1998 (Anderson et al.). Rituximab is
indicated for the treatment of patients with relapsed or refractory
low-grade or follicular, CD20-positive, B cell non-Hodgkin's
lymphoma. In vitro mechanism of action studies have demonstrated
that rituximab binds human complement and lyses lymphoid B cell
lines through complement-dependent cytotoxicity (CDC) (Reff. et.
al., Blood 83(2):435-445 (1994)). Additionally, it has significant
activity in assays for antibody-dependent cellular cytotoxicity
(ADCC). More recently, rituximab has been shown to have
anti-proliferative effects in tritiated thymidine incorporation
assays and to induce apoptosis directly, while other anti-CD19 and
CD20 antibodies do not (Maloney et al., Blood 88(10):637a (1996)).
Synergy between rituximab and chemotherapies and toxins has also
been observed experimentally. In particular, rituximab. sensitizes
drug-resistant human B cell lymphoma cell lines to the cytotoxic
effects of doxorubicin, CDDP, VP-1 6, diphtheria toxin and ricin
(Demidem et al., Cancer Chemotherapy & Radiopharmaceuticals
12(3):177-186 (1997)). In vivo preclinical studies have shown that
rituximab depletes B cells from the peripheral blood, lymph nodes,
and hone marrow of cynomolgus monkeys, presumably through
complement and cell-mediated processes (Reff et al., Blood
83(2):435-445 (1994)).
[0003] 2H7 (ocrelizumab) is a second generation humanized
monoclonal antibody directed against the CD20 surface antigen on
human B-cells. Ocrelizumab is currently tested in Phase III
clinical trials for the treatment of rheumatoid arthritis (RA).
[0004] Patents and patent publications concerning CD20 antibodies
include U.S. Pat. Nos. 5,776,456, 5,736,137, 6,399,061, and
5,843,439, as well as U.S. patent application Nos. US
2002/0197255A1, US 2003/0021781A1, US 2003/0082172 A1, US
2003/0095963 A1, US 2003/0147885 A1 (Anderson et al.); U.S. Pat.
No. 6,455,043B1 and WO00/09160 (Grillo-Lopez, A.); WO00/27428
(Grillo-Lopez and White); WO00/27433 (Grillo-Lopez and Leonard);
WO00/44788 (Braslawsky et al.); WO01/10462 (Rastetter, W.);
WO01/10461 (Rastetter and White); WO01/10460 (White and
Grillo-Lopez); U.S. application No. US2002/0006404 and WO02/04021
(Hanna and Hariharan); U.S. application No. US2002/0012665 A1 and
WO01/74388 (Hanna, N.); U.S. application No. US 2002/0058029 A1
(Hanna, N.); U.S. application No. US 2003/0103971 A1 (Hariharan and
Hanna); U.S. application No. US2002/0009444A1, and WO01/80884
(Grillo-Lopez, A.); WO01/97858 (White, C.); U.S. application No.
US2002/0128488A1 and WO02/34790 (Reff, M.); W)02/060955 (Braslawsky
et al.); WO2/096948 (Braslawsky et al.); WO02/079255 (Reff and
Davies); U.S. Pat. No. 6,171,586B1, and WO98/56418 (Lam et al.);
WO98/58964 (Raju, S.); WO99/22764 (Raju, S.); WO99/51642, U.S. Pat.
No. 6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No.
6,528,624B1 and U.S. Pat. No. 6,538,124 (Idusogie et al.);
WO00/42072 (Presta, L.); WO00/67796 (Curd et al.); WO01/03734
(Grillo-Lopez et al.); U.S. application No. US 2002/0004587A1 and
WO01/77342 (Miller and Presta); U.S. application No. US2002/0197256
(Grewal, I.); U.S. application No. US 2003/0157108 A1 (Presta, L.);
U.S. Pat. Nos. 6,090,365B1, 6,287,537B1, 6,015,542, 5,843,398, and
5,595,721, (Kaminski et al.); U.S. Pat. Nos. 5,500,362, 5,677,180,
5,721,108, and 6,120,767 (Robinson et al.); U.S. Pat. No.
6,410,391B1 (Raubitschek et al.); U.S. Pat. No. 6,224,866B1 and
WO00/20864 (Barbera-Guillem, B.); WO01/13945 (Barbera-Guillem, E.);
WO00/67795 (Goldenberg); U.S. application No. US 2003/01339301 A1
and WO00/74718 (Goldenberg and Hansen); WO00/76542 (Golay et al.);
WO01/72333 (Wolin and Rosenblatt); U.S. Pat. No. 6,368,596B1
(Ghetie et al.); U.S. application No. US2002/0041847 A1,
(Goldenberg, D.); U.S. application No. US2003/0026801A1 (Weiner and
Hartmann); WO02/102312 (Engleman, E.); U.S. patent application No.
2003/0068664 (Albitar et al.); WO03/002607 (Leung, S.); WO
03/049694 and US 2003/0185796 A1 (Wolin et al.); WO03/061694 (Sing
and Siegall); US 2003/0219818 A1 (Bohen et al.); US 2003/0219433 A1
and WO 03/068821 (Hansen et al.), US 2006/0246004 (Adams et
al.);U.S. Pat. No. 5,849,898 and EP application no, 330,191 (Seed
et al.); U.S. Pat. No. 4,861,579 and EP332,865A2 (Meyer and Weiss);
U.S. Pat. No. 4,861,579 (Meyer et al.) and WO95/03770 (Bhat et
al.), each of which is expressly incorporated herein by
reference,
[0005] Publications concerning therapy with Rituximab include:
Perotta and Abuel "Response of chronic relapsing ITP of 10 years
duration to Rituximab" Abstract #3360 Blood 10(1)(part 1-2): p. 88B
(1998); Stashi et al., "Rituximab chimeric anti-CD20 monoclonal
antibody treatment for adults with chronic idopathic
thrombocytopenic purpura" Blood 98(4):952-957 (2001); Matthews, R.
"Medical Heretics" New Scientist (7 Apr., 2001); Leandro et al.,
"Clinical outcome in 22 patients with rheumatoid arthritis treated
with B lymphocyte depletion" Ann Rheum Dis 61:833-888 (2002);
Leandro et al., "Lymphocyte depletion in rheumatoid arthritis:
early evidence for safety, efficacy and dose response. Arthritis
& Rheumatism 44(9): 5370 (2001); Leandro et al., "An open study
of 13 lymphocyte depletion in systemic lupus erythematosus",
Arthritis & Rheumatism 46(1):2673-2677 (2002); Edwards and
Cambridge "Sustained improvement in rheumatoid arthritis following
a protocol designed to deplete B lymphocytes" Rheumatology
40:205-211 (2001); Edwards et al., "B-lymphocyte depletion therapy
in rheumatoid arthritis and other autoimmune disorders" Biochem.
Soc. Trans. 30(4):824-828 (2002); Edwards et al., "Efficacy and
safety of Rituximab, a B-cell targeted chimeric monoclonal
antibody: A randomized, placebo controlled trial in patients with
rheumatoid arthritis. Arthritis & Rheumatism 46(9): S197
(2002); Levine and Pestronk "IgM antibody-related polyneuropathies:
B-cell depletion chemotherapy using Rituximab" Neurology 52:
1701-1704 (1999); DeVita et al., "Efficacy of selective B cell
blockade in the treatment of rheumatoid arthritis" Arthritis &
Rheumatism 46:2029-2033 (2002); Hidashida et al., "Treatment of
DMARD-Refractory rheumatoid arthritis with rituximab." Presented at
the Annual Scientific Meeting of the American College of
Rheumatology; October 24-29; New Orleans. La. 2002; Tuscano, J.
"Successful treatment of Infliximab-refractory rheumatoid arthritis
with rituximab" Presented at the Annual Scientific Meeting of the
American College of Rheumatology; October 24-29; New Orleans, La.
2002. Sarwal et al., N. Eng. J. Med. 349(2):125-138 (Jul. 10, 2003)
reports molecular heterogeneity in acute renal allograft rejection
identified by DNA microarray profiling.
[0006] Production of Antibodies in Mammalian Cell Cultures
[0007] Mammalian cells have become the dominant system for the
production of mammalian proteins for clinical applications,
primarily due to their ability to produce properly folded and
assembled heterologous proteins, and their capacity for
post-translational modifications. Chinese hamster ovary (CHO)
cells, and cell lines obtained from various other mammalian
sources, such as, for example, mouse myeloma (NS0), baby hamster
kidney (BHK), human embryonic kidney (HEK-293) and human retinal
cells have been approved by regulatory agencies for the production
of biopharmaceutical products, including therapeutic antibodies. Of
these, Chinese Hamster Ovary Cells (CHO) are among the most
commonly used industrial hosts, which are commonly used for the
production of heterologous proteins. Thus, methods for the
large-scale production of antibodies in CHO, including
dihydrofolate reductase negative (DHFR-) CHO cells, are well known
in the art (see, e.g. Trill et al., Curr. Opin. Biotechnol.
6(5):553-60 (1995)).
[0008] Usually, to begin the production cycle, a small number of
transformed recombinant host cells is allowed to grow in culture
for several days. Once the cells have undergone several rounds of
replication, they are transferred to a larger container where they
are prepared to undergo fermentation. The media in which the cells
are grown and the levels of oxygen. nitrogen and carbon dioxide
that exist during the production cycle may have a significant
impact on the production process. Growth parameters are determined
specifically for each cell line and these parameters are measured
frequently to assure optimal growth and production conditions.
[0009] When the cells grow to sufficient numbers, they are
transferred to large-scale production tanks and grown for a longer
period of time. At this point in the process, the recombinant
protein can be harvested. Typically, the cells are engineered to
secrete the polypeptide into the cell culture media, so the first
step in the purification process is to separate the cells from the
media. Harvesting usually includes centrifugation and filtration to
produce a Harvested Cell Culture Fluid (HCCF). The media is then
subjected to several additional purification steps that remove any
cellular debris, unwanted proteins, salts, minerals or other
undesirable elements. At the end of the purification process, the
recombinant protein is highly pure and is suitable for human
therapeutic use.
[0010] Although this process has been the subject of much study and
improvements over the past several decades, the production of
recombinant proteins, such as antibodies, is still not without
difficulties. The purification steps are often time consuming,
expensive, and introduce additional issues. With current
improvements in antibody titers from manufacturing cell culture,
purification of the antibody now requires chromatography columns of
unwieldy sizes and large amounts of expensive chromatography resin.
The use of Protein A affinity chromatography columns to remove CHO
host cell proteins (CHOP) from CHO cell cultures is known to
involve Protein A leaching, and requires a further purification
step to remove the leached Protein A. In addition, large-scale
production of polypeptides, such as antibodies, requires the use
and handling of large volumes, which adds to the expense, and often
makes it difficult to achieve satisfactory titers. Thus, there is a
need for improved methods for the large-scale purification of
recombinant polypeptides, such as antibodies. In view of their
established therapeutic importance, it would be particularly
desirable to provide an improved process for the purification of
CD20 antibodies, that would allow the reduction of the number of
purification process steps while maintaining comparable yields to
traditional purification schemes using multiple chromatographic
purification steps.
[0011] There have been limited reports on using crystallization as
part of the purification process of heterologous proteins. U.S.
Application Publication No. 2006/0009387 reports that the
Apo2L/TRAIL protein shows a tendency for spontaneous
crystallization under certain conditions, and, based on this
finding, describes a method for the purification of Apo2L/TRAIL
including a crystallization step.
SUMMARY OF THE INVENTION
[0012] The present invention is based, at least in part, on the
surprising finding that, although antibodies, especially
full-length antibodies, are traditionally difficult to crystallize,
CD20 antibodies can be successfully crystallized from Harvested
Cell Culture Fluid (HCCF) of mammalian cell cultures. In
particular, the invention includes the identification of conditions
that allow the formation of CD20 antibody crystals, including
large, uniform, CD20 antibody crystals, from HCCF. Accordingly, the
present invention provides a process for purifying CD20 antibodies
from mammalian cell cultures, including a crystallization step in
the purification scheme. Incorporation of a crystallization step in
the CD20 antibody purification scheme eliminates chromatographic
steps and their inherent limits of scalability while maintaining
comparable yields to traditional purification schemes that use
multiple chromatographic purification steps, without
crystallization. Accordingly, implementing crystallization into the
purification process results in marked time and cost savings,
without compromising efficiency, product yields or product
quality.
[0013] In one aspect, the invention concerns a method of purifying
a CD20 antibody from a mixture, comprising crystallizing the CD20
antibody and recovering the crystalline CD20 antibody from the
mixture.
[0014] In another aspect, the invention concerns a method of
purifying a CD20 antibody from a mixture comprising the steps of
(a) crystallizing the CD20 antibody to yield CD20 antibody
crystals, (b) dissolving the CD20 antibody crystals to obtain a
CD20 antibody solution, (d) subjecting the CD20 antibody solution
to purification on an anion exchange column, and (e) isolating the
CD20 antibody.
[0015] The mixture can be any mixture comprising CD20 antibodies,
such as any composition obtained during the recombinant production
of CD20 antibodies from any eukaryotic or prokaryotic host
cells.
[0016] In a particular embodiment, the mixture is a Harvested Cell
Culture Fluid (HCCF) from mammalian cells, such as Chinese Hamster
Ovary (CHO) cells; the HCCF may be concentrated beyond its original
concentration out of the bioreactor.
[0017] In another embodiment, the purification is performed in the
absence of a Protein A purification step.
[0018] In yet another embodiment, the purification is performed in
the absence of a cation exchange chromatography step.
[0019] In a further embodiment, the purification is performed in
the absence of both a Protein A purification step and a cation
exchange purification step.
[0020] In another embodiment, the purification scheme comprises a
viral filtration step and an anion exchange purification step,
which are preferably employed subsequent to the to crystallization
purification step.
[0021] In an additional embodiment, the purification method of the
present invention consists essentially of or consists of the
following steps: (a) crystallization of the CD20 antibody from
concentrated HCCF, (b) dissolution of the CD20 crystals in a
buffer, (c) passing the solution obtained through an anion exchange
column, and (d) concentration of the eluate leaving the anion
exchange column.
[0022] In all embodiments, the CD20 antibody can be any diagnostic
or therapeutic CD20 antibody, including, without limitation,
Rituximab (RITUXAN.RTM.), humanized anti-CD20 antibodies including
humanized 2H7 and 2H7 variants, HuMaX-CD20 (Genmab), IMMU-106 (also
known as veltuzumab or hA20; Immunomedics). Monoclonal antibodies
are preferred, which may be chimeric, humanized or human. In all
aspects, the term CD20 "antibody" or "CD20 binding antibody"
specifically includes full length CD20 binding antibody, and
antigen-binding fragments thereof such as Fab or F(ab').sub.2.
Thus, methods for the crystallization of CD20 binding antibodies
and variants thereof are specifically included herein.
[0023] For example, the CD20 antibody may be selected from the
group consisting of 2H7 CD20 antibody variants A-I listed in Table
1. In a particular embodiment, the CD20 antibody is selected from
the group consisting of 2H7 CD20 antibody variants A, C and H
listed in Table 1, having VL and VH pairs of SEQ ID NOs: 1 and 2;
SEQ ID NOs: 3 and 4; and SEQ ID NOs: 3 and 5, respectively.
[0024] In a different aspect, the invention concerns a method of
purifying a CD20 antibody from concentrated Harvested Cell Culture
Fluid (HCCF) of mammalian cells, comprising the steps of (a)
concentrating the HCCF, (b) diafiltering the HCCF with a high salt
concentration at a pH that inhibits crystallization, (c)
crystallizing the CD20 antibody by raising the pH, (d) dissolving
the CD20 antibody crystals to obtain a CD20 antibody solution, (e)
subjecting the CD20 antibody solution to purification on an anion
exchange column, and (f) recovering the resultant purified CD20
antibody.
[0025] Just as before, exemplary CD20 antibodies may be selected
from the group consisting of 2H7 CD20 antibody variants A-I listed
in Table 1. In a particular embodiment, the CD20 antibody is
selected from the group consisting of 2H7 CD20 antibody variants A,
C and H listed in Table 1, having VL and VH pairs of SEQ ID NOs: 1
and 2; SEQ ID NOs: 3 and 4; and SEQ ID NOs: 3 and 5,
respectively.
[0026] In another aspect, the methods of the present invention
concern the crystallization and purification of antibody-like
molecules comprising a CD20 binding sequence, such as CD20 binding
immunoadhesins comprising a Fc region of an IgG. In one embodiment
the CD20 binding immunoadhesin comprises the variable region of the
humanized 2H7 antibody or one of its variants described in Table
1.
[0027] In all embodiments using HCCF as the starting mixture, the
HCCF may be concentrated so that the CD20 antibody concentration is
at a minimum of about 1.5 mg/ml. A CD20 antibody concentration of
about 15 mg/ml provides good yield of antibody crystals with
clearance of CHOP (CHO cell protein) but we have been able to
obtain crystallization of the CD20 binding antibody at as low a
concentration as 1.5 mg/ml.
[0028] In all embodiments, crystallization may be performed in a
wide range of pH, such as, for example, at a pH of about 6.0 to
about 8.0, or of 7.8+/-0.2.
[0029] Crystallization can be preformed in a wide concentration
range, such as, for example at a temperature of about 4.degree. C.
to about 40.degree. C., e.g. at a temperature of about 37.degree.
C.
[0030] Crystallization may be induced by one or more precipitants,
such as one or more precipitants selected from the group consisting
of PBS, NaCl, Na.sub.2SO.sub.4, KCl, K.sub.2SO.sub.4,
Na.sub.2HPO.sub.4, and KH.sub.2PO.sub.4, in particular
KH.sub.2PO.sub.4.
[0031] Crystallization is easier to achieve at higher protein
concentrations but for practical reasons, the HCCF is not
concentrated to a great extent.
[0032] In another aspect, the invention concerns a crystal of a
CD20 antibody. The crystal may be present in different shapes,
including, without limitation microneedle, needle, globular or
globular peanut-shaped crystals, which can be present individually
or in the form of various mixtures, in the presence or absence of
an amorphous, non-crystalline precipitate.
[0033] In a further aspect, the invention concerns a composition
comprising CD20 binding antibody crystals. The composition may, for
example, be a pharmaceutical composition, comprising one or more
pharmaceutically acceptable excipients.
[0034] The invention further concerns a method for treating a B
cell malignancy or an autoimmune disease comprising administering
to a mammalian subject an effective amount of a CD20 antibody
purified by a method of the present invention. In specific
embodiments, the autoimmune disease is selected from the group
consisting of rheumatoid arthritis and juvenile rheumatoid
arthritis, systemic lupus erythematosus (SLE) including lupus
nephritis, Wegener's disease, inflammatory bowel disease,
ulcerative colitis, idiopathic thrombocytopenic purpura (ITP),
thrombotic thrombocytopenic purpura (TTP), autoimmune
thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy,
IgM polyneuropathies, myasthenia gravis, ANCA associated
vasculitis, diabetes mellitus, Reynaud's syndrome, Sjogren's
syndrome, and Neuromyelitis Optica (NMO).
[0035] These and further embodiments will be apparent from the
Examples provided below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is a microscopic view observation of a precipitate
obtained from drug product containing 150 mg/ml 2H7 antibody
dialyzed into beakers containing 20.times.PBS at 37.degree. C., as
described in Example 1.
[0037] FIG. 2 is a microscopic view of large "haystack" 2H7
antibody crystals grown from a solution containing 6 mg/ml 2H7 and
10.times.PBS at 24.degree. C., as described in Example 2.
[0038] FIG. 3 is a microscopic view of needle-shaped and globular
2H7 antibody crystals grown from a solution containing 37.5 mg/ml
2H7 and 10.times.PBS at 37.degree. C., as described in Example
2.
[0039] FIG. 4 is a microscopic view of thin needle 2H7 antibody
crystals obtained grown from a solution containing 5 mg/ml 2H7 and
1 PBS at 4.degree. C., as described in Example 2.
[0040] FIG. 5 is a microscopic view of large round ball-shaped and
needle-shaped 2H7 antibody crystals grown from a solution
containing 37.5 mg/ml 2H7 and 10.times.PBS at 37.degree. C., as
described in Example 3.
[0041] FIG. 6 is a microscopic view of thin. needle-shaped 2H7
antibody crystals obtained from a solution containing 5 mg/ml 2H7
and 10.times.PBS at 37.degree. C., as described in Example 3.
[0042] FIG. 7 is a microscopic view of microneedles of 2H7 antibody
crystals grown from a solution containing 75 mg/ml 2H7 and 300 mM
Na.sub.2HPO.sub.4 at 37.degree. C., as described in Example 3.
[0043] FIG. 8 is a microscopic view of large globular and
peanut-shaped 2H7 antibody crystals obtained from a solution
containing 17.5 mg/ml 2H7 and 500 mM KH.sub.2PO.sub.4 at 37.degree.
C., as described in Example 3.
[0044] FIG. 9 is a microscopic view of globular peanut shaped 2H7
antibody crystals grown from a solution containing 37.5 mg/ml 2H7
and 500 mM KH.sub.2PO.sub.4 at 37.degree. C., as described in
Example 3.
[0045] FIGS. 10A-H show microscopic views of 2H7 antibody crystals
precipitated from a concentrated solution obtained from a
conditioned pool of 2H7 that had been run through a Q-Sepharose
chromatography step (hereinafter referred to as "Q-Pool")
containing 75 mg/ml, 37.5 mg/ml, 17.5 mg/ml or 5 mg/ml 2H7, using
10.times.PBS as a precipitant, in the presence (A, C, E, G) and
absence (B, D, F, H) of Tween/Trehalose.
[0046] FIGS. 11A-C show microscopic views of 2H7 antibody crystals
obtained from a Q-Pool containing 75 mg/ml, 37.5 mg/ml, or 17.5
mg/ml 2H7, using 1M KH.sub.2PO.sub.4 as a precipitant, in the
presence (A, B, D) or absence (C, E) of Tween/Trehalose.
[0047] FIGS. 12A-B graphically summarize the effects of Trehalose
and Tween on crystallization efficiency.
[0048] FIGS. 13A-H show microscopic views of 2H7 antibody crystals
obtained from Harvested Cell Culture Fluid (HCCF) containing 15.5
mg/ml 2H7 in the presence of 10.times.PBS, 15.times.PBS, 500 mM
KH.sub.2PO.sub.4, and 750 mM KH.sub.2PO.sub.4, respectively, using
Q-Pool containing 15.5 mg/ml 2H7 as a control.
[0049] FIG. 14 is a graphical representation of the pH-dependence
of crystallization efficiency from HCCF, using 500 mM
KH.sub.2PO.sub.4 as the precipitant. The 2H7 concentration varied
from 3 mg/ml to 15.5 mg/ml.
[0050] FIG. 15 shows HCCF dissolubility curves at 37.degree. C., at
one hour and 18 hours.
[0051] FIG. 16 shows HCCF dissolubility curves at 24.degree. C., at
one hour and 18 hours.
[0052] FIG. 17 shows HCCF dissolubility curves at 4.degree. C., at
one hour and 18 hours.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0053] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length or
intact monoclonal antibodies), polyclonal antibodies, multivalent
antibodies, multispecific antibodies (e.g., bispecific antibodies)
formed from at least two intact antibodies, and antibody fragments
(see below) so long as they exhibit the desired biological
activity.
[0054] "Antibody fragments" comprise only a portion of an intact
antibody, generally including an antigen binding site of the intact
antibody and thus retaining the ability to bind antigen. Examples
of antibody fragments encompassed by the present definition
include: (i) the Fab fragment, having VL, CL, VH and CH1 domains;
(ii) the Fab' fragment, which is a Fab fragment having one or more
cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd
fragment having VH and CH1 domains; (iv) the Fd' fragment having VH
and CH1 domains and one or more cysteine residues at the C-terminus
of the CH1 domain; (v) the Fv fragment having the VL and VH domains
of a single arm of an antibody; (vi) the dAb fragment (Ward et al.,
Nature 341, 544-546 (1989)) which consists of a VH domain; (vii)
isolated CDR regions; (viii) F(ab').sub.2 fragments, a bivalent
fragment including two Fab' fragments linked by a disulphide bridge
at the hinge region; (ix) single chain antibody molecules (e.g.
single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988);
and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies"
with two antigen binding sites, comprising a heavy chain variable
domain (VH) connected to a light chain variable domain (VL) in the
same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993));
(xi) "linear antibodies" comprising a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions (Zapata et al.
Protein Eng. 8(10):1057 1062 (1995); and U.S. Pat. No.
5,641,870).
[0055] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
Monoclonal antibodies are highly specific, being directed against a
single antigen. In certain embodiments, a monoclonal antibody
typically includes an antibody comprising a polypeptide sequence
that binds a target, wherein the target-binding polypeptide
sequence was obtained by a process that includes the selection of a
single target binding polypeptide sequence from a plurality of
polypeptide sequences. For example, the selection process can be
the selection of a unique clone from a plurality of clones, such as
a pool of hybridoma clones, phage clones, or recombinant DNA
clones. It should be understood that a selected target binding
sequence can be further altered, for example, to improve affinity
for the target, to humanize the target binding sequence, to improve
its production in cell culture, to reduce its immunogenicity in
vivo, to create a multispecific antibody, etc., and that an
antibody comprising the altered target binding sequence is also a
monoclonal antibody of this invention. In contrast to polyclonal
antibody preparations that typically include different antibodies
directed against different determinants (epitopes), each monoclonal
antibody is directed against a single determinant on the antigen.
In addition to their specificity, monoclonal antibody preparations
are advantageous in that they are typically uncontaminated by other
immunoglobulins.
[0056] The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),
phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1991); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132 (2004), and technologies for
producing human or human-like antibodies in animals that have parts
or all of the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol, 7:33 (1993); U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg
et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813
(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);
Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and
Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0057] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA 81:6851-6855 (1984)).
[0058] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also,
e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol.
1:105-115 (1998); Harris, Biochem, Soc. Transactions 23:1035-1038
(1995); Hurle and Gross, Curr. Op. Biotech. 5; 428-433 (1994); and
U.S. Pat. Nos. 6,982,321 and 7,087,409. See also van Dijk and van
de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human
antibodies can be prepared by administering the antigen to a
transgenic animal that has been modified to produce such antibodies
in response to antigenic challenge, but whose endogenous loci have
been disabled. e.g., immunized xenomice (see, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 regarding XENOMOUSE.TM. technology). See
also, for example, Li et al., Proc. Natl. Acad. Sci. USA.
103:3557-3562 (2006) regarding human antibodies generated via a
human B-cell hybridoma technology.
[0059] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art. In one
embodiment, the human antibody is selected from a phage library,
where that phage library expresses human antibodies (Vaughan et al.
Nature Biotechnology 14:309-314 (1996): Sheets et al. PNAS (USA)
95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381
(1991); Marks et al., J Mol. Biol., 222:581 (1991)). Human
antibodies can also be made by introducing human immunoglobulin
loci into transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the
following scientific publications: Marks et al., Bio/Technology 10:
779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994);
Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature
Biotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology
14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93
(1995). Alternatively, the human antibody may be prepared via
immortalization of human B lymphocytes producing an antibody
directed against a target antigen (such B lymphocytes may be
recovered from an individual or may have been immunized in vitro).
See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p, 77 (1985); Boerner et al., J. Immunol., 147
(1):86-95 (1991); and U.S. Pat. No. 5,750,373.
[0060] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a beta-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The hypervariable
regions in each chain are held together in close proximity by the
FRs and, with the hypervariable regions from the other chain,
contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody-dependent cellular toxicity.
[0061] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the amino acid residues of an antibody which are
responsible for antigen-binding. For example, the term
hypervariable region refers to the regions of an antibody variable
domain which are hypervariable in sequence and/or form structurally
defined loops. Generally, antibodies comprise six HVRs; three in
the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., tamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0062] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0063] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0064] "Framework Region" or "FR" residues are those variable
domain residues other than the hypervariable region residues as
herein defined.
[0065] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., supra. Using this numbering system,
the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of 112 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0066] Throughout the present specification and claims, the Kabat
numbering system is generally used when referring to a residue in
the variable domain (approximately, residues 1-107 of the light
chain and residues 1-113 of the heavy chain) (e.g, Kabat et al.,
Sequences of Immunological Interest. 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). The "EU
numbering system" or "EU index" is generally used when referring to
a residue in an immunoglobulin heavy chain constant region (e.g.,
the EU index reported in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991) expressly incorporated
herein by reference). Unless stated otherwise herein, references to
residues numbers in the variable domain of antibodies means residue
numbering by the Kabat numbering system. Unless stated otherwise
herein, references to residue numbers in the constant domain of
antibodies means residue numbering by the EU numbering system
(e.g., see U.S. Provisional Application No. 60/640,323, Figures for
EU numbering).
[0067] Depending on the amino acid sequences of the constant
domains of their heavy chains, antibodies (immunoglobulins) can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG.sub.1
(including non-A and A allotypes), IgG.sub.2, IgG.sub.3, IgG.sub.4,
IgA.sub.1, and IgA.sub.2. The heavy chain constant domains that
correspond to the different classes of immunoglobulins are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known and described
generally in, for example, Abbas et al. Cellular and Mol.
Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody may be
part of a larger fusion molecule, formed by covalent or
non-covalent association of the antibody with one or more other
proteins or peptides.
[0068] The term "Fc region" is used to define the C-terminal region
of an immunoglobulin heavy chain which may be generated by papain
digestion of an intact antibody. The Fc region may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at about position Cys226, or from about
position Pro230, to the carboxyl-terminus of the Fc region. The
C-terminal lysine (residue 447 according to the EU numbering
system) of the Fc region may be removed, for example, during
production or purification of the antibody, or by recombinantly
engineering the nucleic acid encoding a heavy chain of the
antibody. Accordingly, a composition of intact antibodies may
comprise antibody populations with all K447 residues removed,
antibody populations with no K447 residues removed, and antibody
populations having a mixture of antibodies with and without the
K447 residue. The Fc region of an immunoglobulin generally
comprises two constant domains, a CH2 domain and a CH3 domain, and
optionally comprises a CH4 domain.
[0069] Unless indicated otherwise herein, the numbering of the
residues in an immunoglobulin heavy chain is that of the HU index
as in Kabat et al., supra. The "EU index as in Kabat" refers to the
residue numbering of the human IgG1 EU antibody.
[0070] The "CH2 domain" of a human IgG Fc region (also referred to
as "Cg2" domain) usually extends from an amino acid residue at
about position 231 to an amino acid residue at about position 340.
The CH2 domain is unique in that it is not closely paired with
another domain. Rather, two N-linked branched carbohydrate chains
are interposed between the two CH2 domains of an intact native IgG
molecule. It has been speculated that the carbohydrate may provide
a substitute for the domain-domain pairing and help stabilize the
CH2 domain. Burton, Molec. Immunol. 22:161-206 (1985). The CH2
domain herein may be a native sequence CH2 domain or variant CH2
domain.
[0071] The "CH3 domain" comprises the stretch of residues
C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid
residue at about position 341 to an amino acid residue at about
position 447 of an IgG). The CH3 region herein may be a native
sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with
an introduced "protroberance" in one chain thereof and a
corresponding introduced "cavity" in the other chain thereof; see
U.S. Pat. No. 5,821,333, expressly incorporated herein by
reference). Such variant CH3 domains may be used to make
multispecific (e.g. bispecific) antibodies as herein described.
[0072] "Hinge region" is generally defined as stretching from about
Glu216, or about Cys226, to about Pro230 of human IgG1 (Burton,
Molec. Immunol, 22:161-206 (1985)). Hinge regions of other IgG
isotypes may be aligned with the IgG1 sequence by placing the first
and last cysteine residues forming inter-heavy chain S--S bonds in
the same positions. The hinge region herein may be a native
sequence hinge region or a variant hinge region. The two
polypeptide chains of a variant hinge region generally retain at
least one cysteine residue per polypeptide chain, so that the two
polypeptide chains of the variant hinge region can form a disulfide
bond between the two chains. The preferred hinge region herein is a
native sequence human hinge region, e.g. a native sequence human
IgG1 hinge region.
[0073] A "functional Fc region" possesses at least one "effector
function" of a native sequence Fc region. Exemplary "effector
functions" include C1q binding; complement dependent cytotoxicity
(CDC); Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g. B cell receptor; BCR), etc. Such effector functions
generally require the Fc region to be combined with a binding
domain (e.g. an antibody variable domain) and can be assessed using
various assays known in the art for evaluating such antibody
effector functions.
[0074] An "intact" antibody is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (C.sub.L) and heavy chain constant domains, C.sub.H1,
C.sub.H2 and C.sub.H3. The constant domains may be native sequence
constant domains (e.g. human native sequence constant domains) or
amino acid sequence variant thereof. Preferably, the intact
antibody has one or more effector functions.
[0075] A "parent antibody" or "wild-type" antibody is an antibody
comprising an amino acid sequence which lacks one or more amino
acid sequence alterations compared to an antibody variant as herein
disclosed. Thus, the parent antibody generally has at least one
hypervariable region which differs in amino acid sequence from the
amino acid sequence of the corresponding hypervariable region of an
antibody variant as herein disclosed. The parent polypeptide may
comprise a native sequence (i.e. a naturally occurring) antibody
(including a naturally occurring allelic variant), or an antibody
with pre-existing amino acid sequence modifications (such as
insertions, deletions and/or other alterations) of a naturally
occurring sequence. Throughout the disclosure, "wild type," "WT,"
"wt," and "parent" or "parental" antibody are used
interchangeably.
[0076] As used herein, "antibody variant" or "variant antibody"
refers to an antibody which has an amino acid sequence which
differs from the amino acid sequence of a parent antibody. In
certain embodiments, the antibody variant will have an amino acid
sequence from about 75% to less than 100% amino acid sequence
identity or similarity with the amino acid sequence of either the
heavy or light chain variable domain of the parent antibody, more
preferably from about 80% to less than 100%, more preferably from
about 85% to less than 100%. more preferably from about 90% to less
than 100%, and most preferably from about 95% to less than 100%.
The antibody variant is generally one which comprises one or more
amino acid alterations in or adjacent to one or more hypervariable
regions thereof.
[0077] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification. In certain embodiments, the
variant Fc region has at least one amino acid substitution compared
to a native sequence Fc region or to the Fc region of a parent
polypeptide, e.g. from about one to about ten amino acid
substitutions, and preferably from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide, e.g. from about one to about ten
amino acid substitutions, and preferably from about one to about
live amino acid substitutions in a native sequence Fc region or in
the Fc region of the parent polypeptide. The variant Fc region
herein will typically possess, e.g., at least about 80% sequence
identity with a native sequence Fc region and/or with an Fc region
of a parent polypeptide, or at least about 90% sequence identity
therewith, or at least about 95% sequence or more identity
therewith.
[0078] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity (CDC); Fc receptor binding; antibody-dependent
cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of
cell surface receptors (e.g. B cell receptor); and B cell
activation.
[0079] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g. Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The primary cells for mediating ADCC, NK cells, express
Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
F.gamma.RII and Fc.gamma.RIII. FcR expression on hematopoietic
cells is summarized in Table 3 on page 464 of Ravetch and Kinet,
Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a
molecule of interest, an in vitro ADCC assay, such as that
described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed.
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
[0080] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass), which are hound to their cognate
antigen. To assess complement activation, a CDC assay, e.g., as
described in Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996), may be performed. Polypeptide variants with altered Fc
region amino acid sequences (polypeptides with a variant Fc region)
and increased or decreased C1q binding capability are described,
e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642. See also,
e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0081] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result an improvement
in the affinity of the antibody for antigen, compared to a parent
antibody which does not possess those alteration(s). In one
embodiment, an affinity matured antibody has nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al. Bio/Technology 10:779-783 (1992) describes affinity maturation
by VI-1 and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al. Proc Nat. Acad.
Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol.
Biol. 226:889-896 (1992).
[0082] The term "therapeutic antibody" refers to an antibody that
is used in the treatment of disease. A therapeutic antibody may
have various mechanisms of action. A therapeutic antibody may bind
and neutralize the normal function of a target associated with an
antigen. For example, a monoclonal antibody that blocks the
activity of the of protein needed for the survival of a cancer cell
causes the cell's death. Another therapeutic monoclonal antibody
may bind and activate the normal function of a target associated
with an antigen. For example, a monoclonal antibody can bind to a
protein on a cell and trigger an apoptosis signal. Yet another
monoclonal antibody may bind to a target antigen expressed only on
diseased tissue; conjugation of a toxic payload (effective agent),
such as a chemotherapeutic or radioactive agent, to the monoclonal
antibody can create an agent for specific delivery of the toxic
payload to the diseased tissue, reducing harm to healthy tissue. A
"biologically functional fragment" of a therapeutic antibody will
exhibit at least one if not some or all of the biological functions
attributed to the intact antibody, the function comprising at least
specific binding to the target antigen.
[0083] "Purified" means that a molecule is present in a sample at a
concentration of at least 80-90% by weight of the sample in which
it is contained.
[0084] The protein, including antibodies, which is purified is
preferably essentially pure and desirably essentially homogeneous
(i.e. free from contaminating proteins etc.).
[0085] An "essentially pure" protein means a protein composition
comprising at least about 90% by weight of the protein, based on
total weight of the composition, preferably at least about 95% by
weight.
[0086] An "essentially homogeneous" protein means a protein
composition comprising at least about 99% by weight of protein,
based on total weight of the composition.
[0087] The term "storage-stable" is used to describe a formulation
having a shelf-life acceptable for a product in the distribution
chain of commerce, for instance, at least 12 months at a given
temperature, and preferably, at least 24 months at a given
temperature. Optionally, such a storage-stable formulation contains
no more than 5% aggregates, no more than 10% dimers, and/or minimal
changes in charge heterogeneity or biological activity. Degradation
pathways for proteins can involve chemical instability (i.e. any
process which involves modification of the protein by bond
formation or cleavage resulting in a new chemical entity) or
physical instability (i.e. changes in the higher order structure of
the protein). Chemical instability can result from, for example,
deamidation, racemization, hydrolysis, oxidation, beta elimination
or disulfide exchange. Physical instability can result from, for
example, denaturation, aggregation, precipitation or adsorption.
The three most common protein degradation pathways are protein
aggregation, deamidation and oxidation. Cleland et al. Critical
Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377
(1993).
[0088] As used herein, "soluble" refers to polypeptides that, when
in aqueous solutions, are completely dissolved, resulting in a
clear to slightly opalescent solution with no visible particulates,
as assessed by visual inspection. A further assay of the turbidity
of the solution (or solubility of the protein) may be made by
measuring UV absorbances at 340 nm to 360 nm with a 1 cm pathlength
cell where turbidity at 20 mg/ml is less than 0.05 absorbance
units.
[0089] "Preservatives" can act to prevent bacteria, viruses, and
fungi from proliferating in the formulation, and anti-oxidants, or
other compounds can function in various ways to preserve the
stability of the formulation. Examples include
octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,
benzalkonium chloride (a mixture of alkylbenzyldimethylammonium
chlorides in which the alkyl groups are long-chain compounds), and
benzethonium chloride. Other types of compounds include aromatic
alcohols such as phenol and benzyl alcohol, alkyl parabens such as
methyl or propyl paraben, and m-cresol. Optionally, such a compound
is phenol or benzyl alcohol. The preservative or other compound
will optionally be included in a liquid or aqueous form of the CD20
antibody formulation, but not usually in a lyophilized form of the
formulation. In the latter case, the preservative or other compound
will typically be present in the water for injection (WFI) or
bacteriostatic water for injection (BWFI) used for
reconstitution.
[0090] A "surfactant" can act to decrease turbidity or denaturation
of a protein in a formulation. Examples of surfactants include
non-ionic surfactant such as a polysorbate, e.g., polysorbates 20,
60, or 80, a poloxamer, e.g., poloxamer 184 or 188, Pluronic
polyols, ethylene/propylene block polymers or any others known to
the art.
[0091] A "biologically functional fragment" of an antibody
comprises only a portion of an intact antibody, wherein the portion
retains at least one, and as many as most or all, of the functions
normally associated with that portion when present in an intact
antibody. In one embodiment, a biologically functional fragment of
an antibody comprises an antigen binding site of the intact
antibody and thus retains the ability to bind antigen. In another
embodiment, a biologically functional fragment of an antibody, for
example one that comprises the Fc region, retains at least one of
the biological functions normally associated with the Fc region
when present in an intact antibody, such as FcRn binding, antibody
half life modulation, ADCC function and complement binding. In one
embodiment, a biologically functional fragment of an antibody is a
monovalent antibody that has an in vivo half life substantially
similar to an intact antibody. For example, such a biologically
functional fragment of an antibody may comprise an antigen binding
arm linked to an Fc sequence capable of conferring in vivo
stability to the fragment.
[0092] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with research, diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
some embodiments, an antibody is purified (1) to greater than 95%
by weight of antibody as determined by, for example, the Lowry
method, and in some embodiments, to greater than 99% by weight; (2)
to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of, for example, a spinning
cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using, for example, Coomassie blue or silver
stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Ordinarily, however,
isolated antibody will be prepared by at least one purification
step.
[0093] The terms "Protein A" and "ProA" are used interchangeably
herein and encompasses Protein A recovered from a native source
thereof, Protein A produced synthetically (e.g. by peptide
synthesis or by recombinant techniques), and variants thereof which
retain the ability to bind proteins which have a C.sub.H2/C.sub.H3
region, such as an Fc region. Protein A can be purchased
commercially from Repligen, Pharmacia and Fermatech. Protein A is
generally immobilized on a solid phase support material. The term
"ProA" also refers to an affinity chromatography resin or column
containing chromatographic solid support matrix to which is
covalently attached Protein A.
[0094] The term "chromatography" refers to the process by which a
solute of interest in a mixture is separated from other solutes in
a mixture as a result of differences in rates at which the
individual solutes of the mixture migrate through a stationary
medium under the influence of a moving phase, or in bind and elute
processes.
[0095] The term "affinity chromatography" and "protein affinity
chromatography" are used interchangeably herein and refer to a
protein separation technique in which a protein of interest or
antibody of interest is reversibly and specifically bound to a
biospecific ligand. Preferably, the biospecific ligand is
covalently attached to a chromatographic solid phase material and
is accessible to the protein of interest in solution as the
solution contacts the chromatographic solid phase material. The
protein of interest (e.g., antibody, enzyme, or receptor protein)
retains its specific binding affinity for the biospecific ligand
(antigen, substrate, cofactor, or hormone, for example) during the
chromatographic steps, while other solutes and/or proteins in the
mixture do not bind appreciably or specifically to the ligand.
Binding of the protein of interest to the immobilized ligand allows
contaminating proteins or protein impurities to be passed through
the chromatographic medium while the protein of interest remains
specifically bound to the immobilized ligand on the solid phase
material. The specifically bound protein of interest is then
removed in active form from the immobilized ligand with low pH,
high pH, high salt, competing ligand, and the like, and passed
through the chromatographic column with the elution buffer, free of
the contaminating proteins or protein impurities that were earlier
allowed to pass through the column. Any component can be used as a
ligand for purifying its respective specific binding protein, e.g.
antibody.
[0096] The terms "non-affinity chromatography" and "non-affinity
purification" refer to a purification process in which affinity
chromatography is not utilized. Non-affinity chromatography
includes chromatographic techniques that rely on non-specific
interactions between a molecule of interest (such as a protein,
e.g. antibody) and a solid phase matrix.
[0097] A "cation exchange resin" refers to a solid phase which is
negatively charged, and which thus has free cations for exchange
with cations in an aqueous solution passed over or through the
solid phase. A negatively charged ligand attached to the solid
phase to form the cation exchange resin may, e.g., be a carboxylate
or sulfonate. Commercially available cation exchange resins include
carboxy-methyl-cellulose, sulphopropyl (SP) immobilized on agarose
(e.g. SP-SEPHAROSE FAST FLOW.TM. or SP-SEPHAROSE HIGH
PERFORMANCE.TM., from GE Healthcare) and sulphonyl immobilized on
agarose (e.g. S-SEPHAROSE FAST FLOW.TM. from GE Healthcare). A
"mixed mode ion exchange resin" refers to a solid phase which is
covalently modified with cationic, anionic, and hydrophobic
moieties, A commercially available mixed mode ion exchange resin is
BAKERBOND ABX.TM. (J. T. Baker, Phillipsburg, N.J.) containing weak
cation exchange groups, a low concentration of anion exchange
groups, and hydrophobic ligands attached to a silica gel solid
phase support matrix.
[0098] The term "anion exchange resin" is used herein to refer to a
solid phase which is positively charged, e.g. having one or more
positively charged ligands, such as quaternary amino groups,
attached thereto. Commercially available anion exchange resins
include DEAE cellulose, QAE SEPHADEX.TM. and Q SEPHAROSE.TM. FAST
FLOW (GE Healthcare).
[0099] A "buffer" is a solution that resists changes in pH by the
action of its acid-base conjugate components. Various buffers which
can be employed depending, for example, on the desired pH of the
buffer are described in Buffers. A Guide for the Preparation and
Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem
Corporation (1975). In one embodiment, the buffer has a pH in the
range from about 2 to about 9, alternatively from about 3 to about
8, alternatively from about 4 to about 7 alternatively from about 5
to about 7. Non-limiting examples of buffers that will control the
pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate,
acetate, citrate, succinate, and ammonium buffers, as well as
combinations of these.
[0100] The "loading buffer" is that which is used to load the
composition comprising the polypeptide molecule of interest and one
or more impurities onto the ion exchange resin. The loading buffer
has a conductivity and/or pH such that the polypeptide molecule of
interest (and generally one or more impurities) is/are bound to the
ion exchange resin or such that the protein of interest flows
through the column while the impurities bind to the resin.
[0101] The "intermediate buffer" is used to elute one or more
impurities from the ion exchange resin, prior to eluting the
polypeptide molecule of interest. The conductivity and/or pH of the
intermediate buffer is/are such that one or more impurity is eluted
from the ion exchange resin, but not significant amounts of the
polypeptide of interest.
[0102] The term "wash buffer" when used herein refers to a buffer
used to wash or re-equilibrate the ion exchange resin, prior to
eluting the polypeptide molecule of interest. Conveniently, the
wash buffer and loading buffer may be the same, but this is not
required.
[0103] The "elution buffer" is used to elute the polypeptide of
interest from the solid phase. The conductivity and/or pH of the
elution buffer is/are such that the polypeptide of interest is
eluted from the ion exchange resin.
[0104] A "regeneration buffer" may be used to regenerate the ion
exchange resin such that it can be re-used. The regeneration buffer
has a conductivity and/or pH as required to remove substantially
all impurities and the polypeptide of interest from the ion
exchange resin.
[0105] The term "substantially similar" or "substantially the
same," as used herein, denotes a sufficiently high degree of
similarity between two numeric values (for example, one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody), such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is, for
example, less than about 50%, less than about 40%, less than about
30%, less than about 20%, and/or less than about 10% as a function
of the reference/comparator value.
[0106] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid,"
which refers to a circular double stranded DNA into which
additional DNA segments may be ligated. Another type of vector is a
phage vector. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors,"
or simply, "expression vectors." In general, expression vectors of
utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector.
[0107] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, preferably digital
UNIX V4.0D. All sequence comparison parameters are set by the
ALIGN-2 program and do not vary.
[0108] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: [0109] 100 times the fraction X/Y
[0110] where X is the number of amino acid residues scored as
identical matches by [0111] the sequence alignment program ALIGN-2
in that program's alignment of A [0112] and B, and [0113] where Y
is the total number of amino acid residues in B. It will be
appreciated that where the length of amino acid sequence A is not
equal to the length of amino acid sequence B, the % amino acid
sequence identity of A to B will not equal the % amino acid
sequence identity of B to A. Unless specifically stated otherwise,
all % amino acid sequence identity values used herein are obtained
as described in the immediately preceding paragraph using the
ALIGN-2 computer program.
[0114] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented, "Treatment" herein encompasses
alleviation of the disease and of the signs and symptoms of the
particular disease.
[0115] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, non-human higher
primates, other vertebrates, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, horses, cats, cows, etc.
Preferably, the mammal is human.
B. Exemplary Methods and Materials for Carrying Out the
Invention
[0116] The present invention provides CD20 antibody crystals and
methods for recovery and purification of CD20 antibodies. In
particular, the invention provides methods, involving
crystallization, to recover and purify CD20 antibodies from
mixtures in which it is accompanied by other contaminants, such as
contaminating proteins and/or other impurities. In a specific
embodiment, the invention provides methods to recover and purify
CD20 antibodies from recombinant host cultures or cell lysates,
such as mammalian cell cultures or cell lysates of CD20 antibody
producing E. coli recombinant host cells.
[0117] The basis for these purification methods is the
identification of conditions under which CD20 antibodies, including
antibody fragments, readily crystallize in high purity, and in a
size and morphology that allows optimal manipulation throughout the
purification process. It has further been found that the
purification scheme including a crystallization step is well
scaleable, and thus can be used for the large scale purification of
CD20 antibodies.
[0118] The incorporation of a crystallization step in the
purification scheme allows the reduction of purification process
steps while maintaining comparable yields to traditional
purification schemes using multiple chromatographic purification
steps, without crystallization. Accordingly, implementing
crystallization into the purification process results in
significant savings, without compromising efficiency, yields or
product quality.
[0119] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
and the like, which are within the skill of the art. Such
techniques are explained fully in the literature. See e.g.,
Molecular Cloning: A Laboratory Manual, (J. Sambrook et al., Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989); Current
Protocols in Molecular Biology (F. Ausubel et al., eds., 1987
updated); Essential Molecular Biology (T. Brown ed., IRL Press
1991); Gene Expression Technology (Goeddel ed., Academic Press
1991); Methods for Cloning and Analysis of Eukaryotic Genes (A.
Bothwell et al., eds., Bartlett Publ. 1990); Gene Transfer and
Expression (M. Kriegler, Stockton Press 1990); Recombinant DNA
Methodology II (R. Wu et al., eds., Academic Press 1995); PCR: A
Practical Approach (M. McPherson et al., IRL Press at Oxford
University Press 1991); Oligonucleotide Synthesis (M. Gait ed.,
1984); Cell Culture for Biochemists (R. Adams ed., Elsevier Science
Publishers 1990); Gene Transfer Vectors for Mammalian Cells (J.
Miller & M. Calos eds., 1987); Mammalian Cell Biotechnology (M.
Butler ed., 1991); Animal Cell Culture (J. Pollard et al., eds.,
Humana Press 1990); Culture of Animal Cells, 2'' Ed. (R. Freshney
et al., eds., Alan R. Liss 1987); Flow Cytometry and Sorting (M.
Melamed et al., eds., Wiley-Liss 1990); the series Methods in
Enzymology (Academic Press, Inc.); Wirth M. and Hauser H, (1993);
Immunochemistry in Practice, 3rd edition, A. Johnstone & R.
Thorpe, Blackwell Science, Cambridge, Mass., 1996; Techniques in
Immunocytochemistry, (G. Bullock & P. Petrusz eds., Academic
Press 1982, 1983, 1985, 1989); Handbook of Experimental Immunology,
(D. Weir & C. Blackwell, eds.); Current Protocols in Immunology
(J. Coligan et al., eds. 1991); Immunoassay (E. P. Diamandis &
T. K. Christopoulos, eds., Academic Press, Inc., 1996); Goding,
(1986) Monoclonal Antibodies: Principles and Practice (2d ed)
Academic Press, New York; Ed Harlow and David Lane, Antibodies A
laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1988; Antibody Engineering, 2.sup.nd edition (C.
Borrebaeck, ed., Oxford University Press, 1995); and the series
Annual Review of Immunology; the series Advances in Immunology.
[0120] B.1 Production of CD20 Antibodies
[0121] (i) CD20 Antibodies
[0122] In various embodiments, the invention provides crystalline
forms of 2H7 CD20 antibodies, and methods for the purification of
such antibodies, incorporating at least one crystallization step.
In specific embodiments, the humanized 2H7 antibody is an antibody
listed in Table 1.
TABLE-US-00002 TABLE 1 Humanized 2H7 anti-CD20 Antibody and
Variants Thereof V.sub.L V.sub.H Full L chain Full H chain 2H7 SEQ
ID SEQ ID SEQ ID SEQ ID variant NO. NO. NO. NO. A 1 2 6 7 B 1 2 6 8
C 3 4 9 10 D 3 4 9 11 F 3 4 9 12 G 3 4 9 13 H 3 5 9 14 I 1 2 6
15
[0123] Each of the antibody variants A, B and I of Table 1
comprises the light chain variable sequence (V.sub.L):
TABLE-US-00003 (SEQ ID NO: 1)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQG TKVE1KR; and
[0124] the heavy chain variable sequence (V.sub.H):
TABLE-US-00004 (SEQ ID NO: 2)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSNSYWYFDVWGQGTLVTVSS.
[0125] Each of the antibody variants C, D, F and G of Table 1
comprises the light chain variable sequence (V.sub.L):
TABLE-US-00005 (SEQ ID NO: 3)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQG TKVEIKR, and
[0126] the heavy chain variable sequence (V.sub.H):
TABLE-US-00006 (SEQ ID NO: 4)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSASYWYFDVWGQGTLVTVSS.
[0127] The antibody variant H of Table 1 comprises the light chain
variable sequence (V.sub.L) of SEQ NO:3 (above) and the heavy chain
variable sequence (V.sub.H):
TABLE-US-00007 (SEQ ID NO: 5)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRETISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSYRYWYFDVWGQGTLVTVSS
[0128] Each of the antibody variants A, B and I of Table 1
comprises the full length light chain sequence:
TABLE-US-00008 (SEQ ID NO: 6)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGFDFTLTISSLQPEDFATYYCQQWSFNPPTFGQG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC.
[0129] Variant A of Table 1 comprises the full length heavy chain
sequence:
TABLE-US-00009 (SEQ ID NO: 7)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
LYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLINDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK.
[0130] Variant B of Table 1 comprises the full length heavy chain
sequence:
TABLE-US-00010 (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK.
[0131] Variant I of Table 1 comprises the full length heavy chain
sequence:
TABLE-US-00011 (SEQ ID NO: 15)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSNSYWYFDVWGQGTLVTVSSASTKGPSVPPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVIINAKTKPREE
QYNATYRVVSVLTVLIIQDWLNGKEYKCKVSNAALPAPIAATISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK.
[0132] Each of the antibody variants C. D, F, G and H of Table 1
comprises the full length light chain sequence:
TABLE-US-00012 (SEQ ID NO: 9)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQG
TKVEIKRTVAAPSVFIFITSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC.
[0133] Variant C of Table 1 comprises the full length heavy chain
sequence:
TABLE-US-00013 (SEQ ID N0: 10)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTIITCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK.
[0134] Variant D of Table 1 comprises the full length heavy chain
sequence:
TABLE-US-00014 (SEQ ID NO: 11)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSASYWYFDVWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEATISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMIIEALHNHYTQKSLSLS PGK.
[0135] Variant F of Table 1 comprises the full length heavy chain
sequence:
TABLE-US-00015 (SEQ ID NO: 12)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK.
[0136] Variant G of Table 1 comprises the full length heavy chain
sequence:
TABLE-US-00016 (SEQ ID NO: 13)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRETISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSASYWYFDVWGQGTINTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHWHYTQKSLSLSP GK.
[0137] Variant H of Table 1 comprises the lull length heavy chain
sequence:
TABLE-US-00017 (SEQ ID NO: 14)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSP GK.
[0138] In certain embodiments, the humanized 2H7 antibody further
comprises amino acid alterations in the IgG Fc and exhibits
increased binding affinity for human FcRn over an antibody having
wild-type IgG Fc, by at least 60 fold, at least 70 fold, at least
80 fold, more preferably at least 100 fold, preferably at least 125
fold, even more preferably at least 150 fold to about 170 fold.
[0139] The N-glycosylation site in IgG is at Asn297 in the C.sub.H2
domain. Humanized 2H7 antibody compositions of the present
invention include compositions of any of the preceding humanized
2H7 antibodies having a Fc region, wherein about 80-100% (and
preferably about 90-99%) of the antibody in the composition
comprises a mature core carbohydrate structure which lacks fucose,
attached to the Fc region of the glycoprotein. Such compositions
were demonstrated herein to exhibit a surprising improvement in
binding to Fc(RIIIA(F158), which is not as effective as Fc(RIIIA
(V158) in interacting with human IgG. Fc(RIIIA (F158) is more
common than Fc(RIIIA (V158) in normal, healthy African Americans
and Caucasians. See Lehrnbecher el al., Blood 94:4220 (1999).
Historically, antibodies produced in Chinese Hamster Ovary Cells
(CHO), one of the most commonly used industrial hosts, contain
about 2 to 6% in the population that are nonfucosylated. YB2/0 and
Lec13, however, can produce antibodies with 78 to 98%
nonfucosylated species. Shinkawa et al., J Bio. Chem. 278 (5),
3466-347 (2003), reported that antibodies produced in YB2/0 and
Lec13 cells, which have less FUT8 activity, show significantly
increased ADCC activity in vitro. The production of antibodies with
reduced fucose content are also described in e.g., Li et al.,
(GlycoFi) "Optimization of humanized IgGs in glycoengineered Pichia
pastoris" in Nature Biology online publication 22 Jan. 2006; Niwa
R. et al., Cancer Res. 64(6):2127-2133 (2004); US 2003/0157108
(Presta); U.S. Pat. No. 6,602,684 and US 2003/0175884 (Glycart
Biotechnology); US 2004/0093621, US 2004/0110704, US 2004/0132140
(all of Kyowa Hakko Kogyo).
[0140] A bispecific humanized 2H7 antibody encompasses an antibody
wherein one arm of the antibody has at least the antigen binding
region of the H and/or L chain of a humanized 2H7 antibody of the
invention, and the other arm has V region binding specificity for a
second antigen. In specific embodiments, the second antigen is
selected from the group consisting of CD3, CD64, CD32A, CD16, NKG2D
or other NK activating ligands.
[0141] The invention also includes purification of other CD20
antibodies, including, without limitation, the therapeutic antibody
RITUXAN.RTM. (rituximab), which is in clinical practice for the
treatment of relapsed or refractory, low-grade or follicular,
CD20-positive, B-cell non-Hodgkin's lymphoma (NHL); for the
first-line treatment of diffuse large B-cell, CD20-positive,
non-Hodgkin's lymphoma (DLBCL- a type of NHL) in combination with
CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or
other anthracycline-based chemotherapy regimens; for the first-line
treatment of follicular, CD20-positive, B-cell non-Hodgkin's
lymphoma in combination with CVP (cyclophosphamide, vincristine and
prednisolone) chemotherapy; and for the treatment of low-grade,
CD20-positive, B-cell non-Hodgkin's lymphoma in patients with
stable disease or who achieve a partial or complete response
following first-line treatment with CVP chemotherapy.
[0142] (ii) Antibody production
[0143] Monoclonal antibodies, including the CD20 antibodies herein,
may be made using the hybridoma method first described by Kohler et
al., Nature, 256:495 (1975), or may be made by recombinant DNA
methods (U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse
or other appropriate host animal, such as a hamster or macaque
monkey, is immunized as hereinabove described to elicit lymphocytes
that produce or are capable of producing antibodies that will
specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. Lymphocytes
then are fused with myeloma cells using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)).
[0144] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0145] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al, Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).
[0146] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0147] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0148] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0149] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Recombinant production of antibodies will be described
in more detail below.
[0150] In a further embodiment, antibodies or antibody fragments
can be isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990).
[0151] Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks et al.,
Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0152] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567: Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0153] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0154] (iii) Humanized and Human Antibodies
[0155] A humanized antibody has one or more amino acid residues
introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567) wherein substantially less than an intact
human variable domain has been substituted by the corresponding
sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues
from analogous sites in rodent antibodies.
[0156] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0157] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0158] Alternatively, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and Duchosal et al. Nature 355; 258 (1992). Human
antibodies can also be derived from phage-display libraries
(Hoogenboom et al, J. Mol. Biol., 227:381 (1991); Marks et al, J.
Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech
14:309 (1996)). Generation of human antibodies from antibody phage
display libraries is further described below.
[0159] (iv) Antibody Fragments
[0160] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992) and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). In another embodiment as
described in the example below, the F(ab').sub.2 is formed using
the leucine zipper GCN4 to promote assembly of the F(ab').sub.2
molecule. According to another approach, F(ab').sub.2 fragments can
be isolated directly from recombinant host cell culture. Other
techniques for the production of antibody fragments will be
apparent to the skilled practitioner. In other embodiments, the
antibody of choice is a single chain Fv fragment (scFv). See WO
93/16185.
[0161] (v) Recombinant Production of Antibodies
[0162] For recombinant production of an antibody, the nucleic acid
encoding it is isolated and inserted into a replicable vector for
further cloning (amplification of the DNA) or for expression. DNA
encoding the monoclonal antibody is readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding
the heavy and light chains of the antibody). Many vectors are
available. The vector components generally include, but are not
limited to, one or more of the following: a signal sequence, an
origin of replication, one or more marker genes, an enhancer
element, a promoter, and a transcription termination sequence (e.g.
as described in U.S. Pat. No. 5,534,615, specifically incorporated
herein by reference).
[0163] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubactcria, such as Gram-negative or Gram-positive organisms, for
example. Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhinurium, Serrafia, e.g, Serratia marcescans, and
Shigeila, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli X
1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0164] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors. Saccharomyces cerevisiae, or common
baker's yeast, is the most commonly used among lower eukaryotic
host microorganisms. However, a number of other genera, species,
and strains are commonly available and useful herein, such as
Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0165] Suitable host cells for the expression of glycosylated
antibody are derived from multicellular organisms. Examples of
invertebrate cells include plant and insect cells. Numerous
baculoviral strains and variants and corresponding permissive
insect host cells from hosts such as Spodoptera frugiperda
(caterpillar), Aedes aegypti (mosquito), Aedes albopictus
(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
have been identified. A variety of viral strains for transfection
are publicly available, e.g., the L-1 variant of Autographa
californica NPV and the Bm-5 strain of Bombyx mori NPV, and such
viruses may be used as the virus herein according to the present
invention, particularly for transfection of Spodoptera frugiperda
cells. Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can also be utilized as hosts.
[0166] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed bySV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subloned
for growth in suspension culture, Graham et al, J. Gen Virol. 36:59
(1977)); baby hamster kidney cells (BHK, ATCC CRL 10); Chinese
hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad.
Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);
African green monkey kidney cells (VERO-76, ATCC MA 587); human
cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep
G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI
cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC
5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0167] In one embodiment, the CD20 antibodies herein are produced
in dp12.CHO cells, the production of which from CHO-K1 DUX-B11
cells as described in EP307247. CHO-K1 DUX-B11 cells were, in turn,
obtained from CHO-K1 (ATCC No. CCL61 CHO-K1) cells, following the
methods described in Simonsen, C. C., and Levinson, A. D., (1983)
Proc. Natl. Acad. Sci. USA 80:2495-2499 and Urlaub G., and Chasin,
L., (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, n addition,
other CHO-K1 (dhfr) cell lines are known and can be used in the
methods of the present invention.
[0168] The mammalian host cells used to produce peptides,
polypeptides and proteins can be cultured in a variety of media.
Commercially available media such as Ham's F10 (Sigma), Minimal
Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's
Modified Eagle's Medium ((DMEM, Sigma) are suitable for culturing
the host cells. In addition, any of the media described in Ham and
Wallace (1979), Meth. in Enz. 58:44, Barnes and Sato (1980), Anal.
Biochem. 102:255, U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;
or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. No. Re. 30,985;
or U.S. Pat. No. 5,122,469, the disclosures of all of which are
incorporated herein by reference, may be used as culture media for
the host cells. Any of these media may be supplemented as necessary
with hormones and/or other growth factors (such as insulin,
transferrin, or epidermal growth factor), salts (such as sodium
chloride, calcium, magnesium, and phosphate), buffers (such as
HEPES), nucleosides (such as adenosine and thymidine), antibiotics
(such as Gentamycin.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0169] B.2 Crystallization of C20 Antibodies
[0170] Crystallization is widely used for purification of small
molecules. However, generally, finding crystallization conditions
for proteins, especially for full-length antibodies, where the
proper assembly of the three-dimensional antibody structure raises
special issues, is very difficult and tedious. Parameters affecting
crystallization include, for example, solubility, nucleation and
growth rate, and crystal size distribution, each being a function
of further parameters, such as temperature, pH, buffer, impurities,
and the like. Since antibodies are much more difficult to
crystallize than small molecules or small proteins or proteins of
simpler structure, the recovery and purification of therapeutic
antibodies rarely involves a crystallization step.
[0171] B.3 Use of Crystallization in the Recovery and Purification
of CD20 Antibodies
[0172] In the methods of the present invention, crystallization is
a key step in a one-column or a two-column scheme for the recovery
and purification of CD20 antibodies.
[0173] A protocol for the production, recovery and purification of
recombinant antibodies in mammalian, such as CHO, cells may include
the following steps:
[0174] Cells may be cultured in a stirred tank bioreactor system
and a fed batch culture, procedure is employed. In a preferred fed
batch culture the mammalian host cells and culture medium are
supplied to a culturing vessel initially and additional culture
nutrients are fed, continuously or in discrete increments, to the
culture during culturing, with or without periodic cell and/or
product harvest before termination of culture. The fed batch
culture can include, for example, a semi-continuous fed batch
culture, wherein periodically whole culture (including cells and
medium) is removed and replaced by fresh medium. Fed batch culture
is distinguished from simple batch culture in which all components
for cell culturing (including the cells and all culture nutrients)
are supplied to the culturing vessel at the start of the culturing
process. Fed batch culture can be further distinguished from
perfusion culturing insofar as the supernate is not removed from
the culturing vessel during the process (in perfusion culturing,
the cells are restrained in the culture by, e.g., filtration,
encapsulation, anchoring to microcarriers etc. and the culture
medium is continuously or intermittently introduced and removed
from the culturing vessel).
[0175] Further, the cells of the culture may be propagated
according to any scheme or routine that may be suitable for the
particular host cell and the particular production plan
contemplated. Therefore, a single step or multiple step culture
procedure may be employed. In a single step culture the host cells
are inoculated into a culture environment and the processes are
employed during a single production phase of the cell culture.
Alternatively, a multi-stage culture can be used. In the
multi-stage culture cells may be cultivated in a number of steps or
phases. For instance, cells may be grown in a first step or growth
phase culture wherein cells, possibly removed from storage, are
inoculated into a medium suitable for promoting growth and high
viability. The cells may be maintained in the growth phase for a
suitable period of time by the addition of fresh medium to the host
cell culture.
[0176] In certain embodiments, fed batch or continuous cell culture
conditions may be devised to enhance growth of the mammalian cells
in the growth phase of the cell culture. In the growth phase cells
are grown under conditions and for a period of time that is
maximized for growth. Culture conditions, such as temperature, pH,
dissolved oxygen (dO.sub.2) and the like, are those used with the
particular host and will be apparent to the ordinarily skilled
artisan. Generally, the pH is adjusted to a level between about 6.5
and 7.5 using either an acid (e.g., CO.sub.2) or a base (e.g.,
Na.sub.2CO.sub.3 or NaOH). A suitable temperature range for
culturing mammalian cells such as CHO cells is between about
30.degree. C. to 38.degree. C., and a suitable dO.sub.2 is between
5-90% of air saturation.
[0177] At a particular stage the cells may be used to inoculate a
production phase or step of the cell culture. Alternatively, as
described above the production phase or step may be continuous with
the inoculation or growth phase or step.
[0178] The cell culture environment during the production phase of
the cell culture is typically controlled. Thus, if a glycoprotein
is produced, factors affecting cell specific productivity of the
mammalian host cell may be manipulated such that the desired sialic
acid content is achieved in the resulting glycoprotein. In a
preferred aspect, the production phase of the cell culture process
is preceded by a transition phase of the cell culture in which
parameters for the production phase of the cell culture are
engaged. Further details of this process are found in U.S. Pat. No.
5,721,121, and Chaderjian et al., Biotechnol. Prog. 21(4550-3
(2005), the entire disclosures of which are expressly incorporated
by reference herein.
[0179] Following fermentation proteins are purified. Procedures for
purification of proteins from cell debris initially depend on the
site of expression of the protein. Some proteins can be caused to
be secreted directly from the cell into the surrounding growth
media; others are made intracellularly. For the latter proteins,
the first step of a purification process involves lysis of the
cell, which can be done by a variety of methods, including
mechanical shear, osmotic shock, or enzymatic treatments. Such
disruption releases the entire contents of the cell into the
homogenate, and in addition produces subcellular fragments that are
difficult to remove due to their small size. These are generally
removed by differential centrifugation or by filtration. The same
problem arises, although on a smaller scale, with directly secreted
proteins due to the natural death of cells and release of
intracellular host cell proteins and components in the course of
the protein production run.
[0180] Once a clarified solution containing the protein of interest
has been obtained, its separation from the other proteins produced
by the cell is usually attempted using a combination of different
chromatography techniques. These techniques separate mixtures of
proteins on the basis of their charge, degree of hydrophobicity, or
size. Several different chromatography resins are available for
each of these techniques, allowing accurate tailoring of the
purification scheme to the particular protein involved. The essence
of each of these separation methods is that proteins can be caused
either to move at different rates down a long column, achieving a
physical separation that increases as they pass further down the
column, or to adhere selectively to the separation medium, being
then differentially eluted by different solvents. In some cases,
the desired protein is separated from impurities when the
impurities specifically adhere to the column, and the protein of
interest does not, that is, the protein of interest is present in
the "flow-through." Thus, purification of recombinant proteins from
the cell culture of mammalian host cells may include one or more
affinity (e.g. protein A) and/or ion exchange chomarographic
steps.
[0181] Ion exchange chromatography is a chromatographic technique
that is commonly used for the purification of proteins. In ion
exchange chromatography, charged patches on the surface of the
solute are attracted by opposite charges attached to a
chromatography matrix, provided the ionic strength of the
surrounding buffer is low. Elution is generally achieved by
increasing the ionic strength (i.e., conductivity) of the buffer to
compete with the solute for the charged sites of the ion exchange
matrix. Changing the pH and thereby altering the charge of the
solute is another way to achieve elution of the solute. The change
in conductivity or pH may be gradual (gradient elution) or stepwise
(step elution). In the past, these changes have been progressive;
i.e., the pH or conductivity is increased or decreased in a single
direction.
[0182] For further details of the industrial purification of
therapeutic antibodies sec, for example, Fahrner et al.,
Biotechnol. Genet. Eng. Rev. 18:301-27 (2001), the entire
disclosure of which is expressly incorporated by reference
herein.
[0183] A typical protocol for purifying recombinant proteins, such
as antibodies, from CHO cell cultures includes the following steps:
(1) Protein A chromatography, (2) cation exchange chromatography,
(3) viral filtration, (4) anion exchange chromatography and (5)
ultrafiltration--diafiltration (UFDF).
[0184] Protein A chromatography removes CHO cell proteins (CHOP),
CHO cell DNA, gentamycin, insulin, and inactive viral
contamination.
[0185] Cation exchange chromatography retains biomolecules by the
interaction of charged groups that are acidic in nature on the
surface of the resin with histidine, lysine and arginine. Cation
exchange resins are commercially available from the product lines
of various manufacturers, such as, for example. Sigma Aldrich.
Cation exchangers include resins carrying, for example,
carboxymethyl functional groups (weak cation exchanger, such as, CM
cellulose/SEPHADEX.RTM. or sulfonic acid functional groups (strong
cation exchanger, such as, SP SEPHADEX.RTM.). In the second
chromatographic purification step of the methods of the present
invention, strong cation exchange columns, e.g. SP-SEPHADEX.RTM.,
SPECTRA/GEL.RTM. strong cation exchangers, etc. TSKgel strong
cation exchangers, etc. are preferred. In the case of an
SP-SEPHAROSE.RTM. column, the cross-linked agarose matrix with
negatively charged functional groups binds to the CD20 antibody
while allowing the majority of the impurities to pass through the
column. Elution can be performed using salt gradient elution or
step elution, step elution being preferred since it provides better
conditions for the subsequent crystallization step, without
compromising yields. The elution buffer usually contains sodium
chloride or sodium sulfate, and salt concentration is selected to
meet the demands of the cation exchange column. The
SP-SEPHAROSE.RTM. column needs a fairly high salt concentration to
remove the bound CD20 protein, while for the subsequent
crystallization step relatively low salt concentrations are
preferred, in order to lower protein solubility. Typically, about
100-150 mM Na.sub.2SO.sub.4 or 100-200 mM NaCl concentrations are
used. A typical elution buffer consists of 200 mM NaCl, 50 mM
HEPES, 0.05% Triton X-100, 1 mM DTT, pH 7.5. The cation
chromatography step used to remove the remaining CHOP, leached
Protein A, remaining CHO DNA, gentamycin, insulin and antibody
aggregates.
[0186] The viral filtration step provides for high level retrovirus
clearance.
[0187] Anion exchange chromatography employs resins which are
positively charged, e.g. have one or more positively charged
ligands, such as quaternary amino groups, attached thereto.
Commercially available anion exchange resins include DEAE
cellulose, QAE SEPHADEX.RTM.. and Q SEPHAROSE Fast Flow.RTM. (GE
Healthcare). The anion exchange step removes the final remnants of
CHOP and CHO DNA and viral impurities, and the UFDF step
concentrates and formulates the Q pool.
[0188] The present invention provides a purification scheme, in
which one or more steps of the traditional purification process are
replaced by a crystallization step. Thus, for example, the protein
A and subsequent cation exchange purification steps can be replaced
by a step of concentrating the HCCF followed by crystallization of
the CD20 antibody. The crystallization step effectively removes
CHOP, CHO DNA, gentamycin and insulin. In the process including a
crystallization step, the CHOP and CHO DNA levels are lower than
the corresponding levels after two chromatographic purification
steps. In addition, since a Protein A chromatography step is not
included, there is no need for the removal of leached Protein A,
which results in significant savings. Thus, the new method
described herein for the purification of CD20 antibodies from
recombinant cell cultures yields a reduction in raw materials and
process steps, and yields a highly efficient and scaleable
purification scheme, suitable for the large scale production of
CD20 antibodies.
[0189] While the examples illustrate purification from a mammalian
(CHO) cell culture, a similar approach can be applied for the
purification of CD20 antibodies from bacterial, e.g. E. coli cells.
If the CD20 antibody is produced in E. coli, typically the whole
cell broth is harvested and homogenized to break open the E. coli
cells and release antibody within the cytoplasm. After removing the
solid debris, e.g. by centrifugation, the mixture is loaded onto a
cation exchange chromatographic column, such as, for example,
SP-Sepharose Fast Flow column (Amersham Pharmacia, Sweden).
[0190] In a typical protocol, the pH of the whole cell broth
obtained by fermentation of the E. coli cells is adjusted to about
7.5, e.g. by addition of sodium HEPES or any other appropriate
buffer. The cells are burst open by one or more passes on a
commercially available homogenizer, the cell debris is removed, and
the cell lysate is clarified. Specific treatment parameters, such
as selection and concentration of reagents, depend on the
composition of the starting whole cell broth, such as, for example,
cell density. In this cast, the crystallization step might follow
cation exchange, e.g. SP-SEPHAROSE.RTM. purification. The
concentration must be high enough to maximize the solubility
differences at different temperatures, but not too high to trigger
spontaneous crystallization at or around room temperature.
[0191] When crystallization is complete, the CD20 antibody crystals
are removed, for example by filtration. The crystals may be kept
suspended throughout filtration, using a built-in agitator, or can
be deposited in a packed bed. It is important to avoid the
formation of a compressed crystal cake, which could make it
impossible to achieve the desired flow rate. Flow rates may vary,
and typically are between about 200 cm/hr and about 100 cm/hr. The
flow rate may depend on the equipment used, and the pressure to be
applied during filtration. Filtration may be performed batch-wise
or continuously.
[0192] Following crystallization and separation, the anti-CD20
antibody crystals can be redissolved and stored or converted into a
formulation suitable for the intended use.
[0193] Alternatively, a further chromatography purification step
can be added to further improve purity by removing the anti-solvent
(PEG) residues and buffer components, and reduce the levels of
residual extracellular proteins, endotoxin, dimers, and
aggregates.
[0194] In summary, the purification method for the CD20 binding
antibodies of the present invention involves the steps of
concentrating the HCCF, crystallizing the antibody under the
appropriate conditions, removing and washing the resultant antibody
crystals, redissolving the antibody crystals, subjecting the
antibody solution to a chromatography purification step, e.g.,
Q-Sepharose chromatography, and exchanging the purified antibody
into the desired formulation using, for example,
ultrafiltration/diafiltration.
[0195] B.4 Use of Purified Antibodies in Methods of Treatment
[0196] The CD20 binding antibodies purified by the methods of the
present invention are useful to treat or alleviate an autoimmune
disease or a B cell malignancy either as front line therapy or
after other treatment, or in conjunction with a second therapeutic
agent, either concurrently, sequentially or in alternating regimen.
In preferred embodiments the antibody is administered intravenously
or subcutaneously.
[0197] The methods of treating a CD20 positive, B cell malignancy
comprises administering to a patient having the malignancy, a
therapeutically effective amount of a CD20 antibody purified by the
present methods using crystallization. In specific embodiments, the
CD20 antibody is a humanized 2H7 antibody described in Table 1. In
specific embodiments the B cell malignancy is a B cell lymphoma or
leukemia including non-Hodgkin's lymphoma (NHL), lymphocyte
predominant Hodgkin's disease (LPHD), small lymphocytic lymphoma
(SLL), chronic lymphocytic leukemia (CLL). Where the B-cell
lymphoma is non-Hodgkin's lymphoma (NHL), the NHL includes, but is
not limited to, follicular lymphoma, relapsed follicular lymphoma,
small lymphocytic lymphoma, mantle cell lymphoma, marginal zone
lymphoma, lymphoplasmacytic lymphoma, mycosis fungoides/Sezary
syndrome, splenic marginal zone lymphoma, and diffuse large B-cell
lymphoma. In some embodiments, the B-cell lymphoma is selected from
the group consisting of indolent lymphoma, aggessive lymphoma, and
highly aggressive lymphoma. In specific embodiments, humanized CD20
binding antibodies or functional fragments thereof, are used to
treat indolent NHL including relapsed indolent NHL and
rituximab-refractory indolent NHL.
[0198] An "autoimmune disease" herein is a disease or disorder
arising from and directed against an individual's own tissues or
organs or a co-segregate or manifestation thereof or resulting
condition therefrom. In many of these autoimmune and inflammatory
disorders, a number of clinical and laboratory markers may exist,
including, but not limited to, hypergammaglobulinemia, high levels
of autoantibodies, antigen-antibody complex deposits in tissues,
benefit from corticosteroid or immunosuppressive treatments, and
lymphoid cell aggregates in affected tissues. Without being limited
to any one theory regarding B-cell mediated autoimmune disease, it
is believed that B cells demonstrate a pathogenic effect in human
autoimmune diseases through a multitude of mechanistic pathways,
including autoantibody production, immune complex formation,
dendritic and T-cell activation, cytokine synthesis, direct
chemokine release, and providing a nidus for ectopic
neo-lymphogenesis. Each of these pathways may participate to
different degrees in the pathology of autoimmune diseases.
[0199] "Autoimmune disease" can be an organ-specific disease (i.e.,
the immune response is specifically directed against an organ
system such as the endocrine system, the hematopoietic system, the
skin, the cardiopulmonary system, the gastrointestinal and liver
systems, the renal system, the thyroid, the ears, the neuromuscular
system, the central nervous system, etc.) or a systemic disease
which can affect multiple organ systems (for example, systemic
lupus erythematosus (SLE), rheumatoid arthritis, polymyositis,
etc.). Preferred such diseases include autoimmune rheumatologic
disorders (such as, for example, rheumatoid arthritis, Sjogren's
syndrome, scleroderma, lupus such as SLE and lupus nephritis,
polymyositis/dermatomyositis. cryoglobulinemia, anti-phospholipid
antibody syndrome, and psoriatic arthritis), autoimmune
gastrointestinal and liver disorders (such as, for example,
inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's
disease), autoimmune gastritis and pernicious anemia, autoimmune
hepatitis, primary biliary cirrhosis, primary sclerosing
cholangitis, and celiac disease), vasculitis (such as, for example,
ANCA-negative vasculitis and ANCA-associated vasculitis, including
Churg-Strauss vasculitis, Wegener's granulomatosis, and microscopic
polyangiitis), autoimmune neurological disorders (such as, for
example, multiple sclerosis, opsoclonus myoclonus syndrome,
myasthenia gravis, neuromyelitis optica, Parkinson's disease,
Alzheimer's disease, and autoimmune polyneuropathies), renal
disorders (such as, for example, glomerulonephritis, Goodpasture's
syndrome, and Berger's disease), autoimmune dermatologic disorders
(such as, for example, psoriasis, urticaria, hives, pemphigus
vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus),
hematologic disorders (such as, for example, thrombocytopenic
purpura, thrombotic thrombocytopenic purpura, post-transfusion
purpura, and autoimmune hemolytic anemia), atherosclerosis,
uveitis, autoimmune hearing diseases (such as, for example, inner
car disease and hearing loss), Behcet's disease, Raynaud's
syndrome, organ transplant, and autoimmune endocrine disorders
(such as, for example, diabetic-related autoimmune diseases such as
insulin-dependent diabetes mellitus (IDDM), Addison's disease, and
autoimmune thyroid disease (e.g., Graves' disease and
thyroiditis)). More preferred such diseases include, for example,
rheumatoid arthritis, ulcerative colitis, ANCA-associated
vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves'
disease, IDDM, pernicious anemia, thyroiditis, and
glomerulonephritis.
[0200] Specific examples of other autoimmune diseases as defined
herein, which in some cases encompass those listed above, include,
but are not limited to, arthritis (acute and chronic, rheumatoid
arthritis including juvenile-onset rheumatoid arthritis and stages
such as rheumatoid synovitis, gout or gouty arthritis, acute
immunological arthritis, chronic inflammatory arthritis,
degenerative arthritis, type II collagen-induced arthritis,
infectious arthritis, Lyme arthritis, proliferative arthritis,
psoriatic arthritis, Still's disease, vertebral arthritis,
osteoarthritis, arthritis chronica progrediente, arthritis
deformans, polyarthritis chronica primaria, reactive arthritis,
menopausal arthritis, estrogen-depletion arthritis, and ankylosing
spondylitis/rheumatoid spondylitis), autoimmune lymphoproliferative
disease, inflammatory hyperproliferative skin diseases, psoriasis
such as plaque psoriasis, guttate psoriasis, pustular psoriasis,
and psoriasis of the nails, atopy including atopic diseases such as
hay fever and Job's syndrome, dermatitis including contact
dermatitis, chronic contact dermatitis, exfoliative dermatitis,
allergic dermatitis, allergic contact dermatitis, hives, dermatitis
herpetiformis, nummular dermatitis, seborrheic dermatitis,
non-specific dermatitis, primary irritant contact dermatitis, and
atopic dermatitis, x-linked hyper IgM syndrome, allergic
intraocular inflammatory diseases, urticaria such as chronic
allergic urticaria and chronic idiopathic urticaria, including
chronic autoimmune urticaria, myositis,
polymyositis/dermatomyositis, juvenile dermatomyositis, toxic
epidermal necrolysis, scleroderma (including systemic scleroderma),
sclerosis such as systemic sclerosis, multiple sclerosis (MS) such
as spino-optical MS, primary progressive MS (PPMS), and relapsing
remitting MS (RRMS), progressive systemic sclerosis,
atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic
sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease
(IBD) (for example, Crohn's disease, autoimmune-mediated
gastrointestinal diseases, gastrointestinal inflammation, colitis
such as ulcerative colitis, colitis ulcerosa, microscopic colitis,
collagenous colitis, colitis polyposa, necrotizing enterocolitis,
and transmural colitis, and autoimmune inflammatory bowel disease),
bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary
sclerosing cholangitis, respiratory distress syndrome, including
adult or acute respiratory distress syndrome (ARDS), meningitis,
inflammation of all or part of the uvea, iritis, choroiditis, an
autoimmune hematological disorder, graft-versus-host disease,
angioedema such as hereditary angioedema, cranial nerve damage as
in meningitis, herpes gestationis, pemphigoid gestationis, pruritic
scroti, autoimmune premature ovarian failure, sudden hearing loss
due to an autoimmune condition, IgE-mediated diseases such as
anaphylaxis and allergic and atopic rhinitis, encephalitis such as
Rasmussen's encephalitis and limbic and/or brainstem encephalitis,
uveitis, such as anterior uveitis, acute anterior uveitis,
granulomatous uveitis, nongranulomatous uveitis, phacoantigcnic
uveitis, posterior uveitis, or autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as
chronic or acute glomerulonephritis such as primary GN,
immune-mediated GN, membranous GN (membranous nephropathy),
idiopathic membranous GN or idiopathic membranous nephropathy,
membrano- or membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN (RPGN), proliferative
nephritis, autoimmune polyglandular endocrine failure, balanitis
including balanitis circumscripta plasmacellularis,
balanoposthitis, erythema annulare centrifugum, erythema
dyschromicum perstans, eythema multiform, granuloma annulare,
lichen nitidus, lichen sclerosus et atrophicus, lichen simplex
chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis,
epidermolytic hyperkeratosis, premalignant keratosis, pyoderma
gangrenosum, allergic conditions and responses, food allergies,
drug allergies, insect allergies, rare allergic disorders such as
mastocytosis, allergic reaction, eczema including allergic or
atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular
palmoplantar eczema, asthma such as asthma bronchiale, bronchial
asthma, and auto-immune asthma, conditions involving infiltration
of T cells and chronic inflammatory responses, immune reactions
against foreign antigens such as fetal A-B-O blood groups during
pregnancy, chronic pulmonary inflammatory disease, autoimmune
myocarditis, leukocyte adhesion deficiency, lupus, including lupus
nephritis, lupus cerebritis, pediatric lupus, non-renal lupus,
extra-renal lupus, discoid lupus and discoid lupus erythematosus,
alopecia lupus, SLE, such as cutaneous SLE or subacute cutaneous
SLE, neonatal lupus syndrome (NLE), and lupus erythematosus
disseminatus, juvenile onset (Type I) diabetes mellitus, including
pediatric IDDM, adult onset diabetes mellitus (Type II diabetes),
autoimmune diabetes, idiopathic diabetes insipidus, diabetic
retinopathy, diabetic nephropathy, diabetic colitis, diabetic
large-artery disorder, immune responses associated with acute and
delayed hypersensitivity mediated by cytokines and T-lymphocytes,
tuberculosis, sarcoidosis, granulomatosis including lymphomatoid
granulomatosis, agranulocytosis, vasculitides (including
large-vessel vasculitis such as polymyalgia rheumatica and
giant-cell (Takayasu's) arteritis, medium-vessel vasculitis such as
Kawasaki's disease and polyarteritis nodosa/periarteritis nodosa,
immunovasculitis, CNS vasculitis, cutaneous vasculitis,
hypersensitivity vasculitis, necrotizing vasculitis such as
fibrinoid necrotizing vasculitis and systemic necrotizing
vasculitis, ANCA-negative vasculitis, and ANCA-associated
vasculitis such as Churg-Strauss syndrome (CSS), Wegener's
granulomatosis, and microscopic polyangiitis), temporal arteritis,
aplastic anemia, autoimmune aplastic anemia, Coombs positive
anemia, Diamond Blackfan anemia, hemolytic anemia or immune
hemolytic anemia including autoimmune hemolytic anemia (AIHA),
pernicious anemia (anemia perniciosa), Addison's disease, pure red
cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia
A, autoimmune neutropenia(s), cytopenias such as pancytopenia,
leukopenia, diseases involving leukocyte diapedesis, CNS
inflammatory disorders, Alzheimer's disease, Parkinson's disease,
multiple organ injury syndrome such as those secondary to
septicemia, trauma or hemorrhage, antigen-antibody complex-mediated
diseases, anti-glomerular basement membrane disease,
anti-phospholipid antibody syndrome, motoneuritis, allergic
neuritis, Behcet's disease/syndrome, Castleman's syndrome,
Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome,
Stevens-Johnson syndrome, pemphigoid or pemphigus such as
pemphigoid bullous, cicatricial (mucous membrane) pemphigoid, skin
pemphigoid, pemphigus vulgaris, paraneoplastic pemphigus, pemphigus
foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus
erythematosus, epidermolysis bullosa acquisita, ocular
inflammation, preferably allergic ocular inflammation such as
allergic conjunctivis, linear IgA bullous disease,
autoimmune-induced conjunctival inflammation, autoimmune
polyendocrinopathies, Reiter's disease or syndrome, thermal injury
due to an autoimmune condition, preeclampsia, an immune complex
disorder such as immune complex nephritis, antibody-mediated
nephritis, neuroinflammatory disorders, polyneuropathies, chronic
neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy,
thrombocytopenia (as developed by myocardial infarction patients,
for example), including thrombotic thrombocytopenic purpura (TTP),
post-transfusion purpura (PTP), heparin-induced thrombocytopenia,
and autoimmune or immune-mediated thrombocytopenia including, for
example, idiopathic thrombocytopenic purpura (ITP) including
chronic or acute ITP, scleritis such as idiopathic
cerato-scleritis, episcleritis, autoimmune disease of the testis
and ovary including autoimmune orchitis and oophoritis, primary
hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases
including thyroiditis such as autoimmune thyroiditis, Hashimoto's
disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute
thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,
Grave's disease, Grave's eye disease (opthalmopathy or
thyroid-associated opthalmopathy), polyglandular syndromes such as
autoimmune polyglandular syndromes, for example, type I (or
polyglandular endocrinopathy syndromes), paraneoplastic syndromes,
including neurologic paraneoplastic syndromes such as Lambert-Eaton
myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or
stiff-person syndrome, encephalomyelitis such as allergic
encephalomyelitis or encephalomyelitis allergica and experimental
allergic encephalomyelitis (EAE), myasthenia gravis such as
thymoma-associated myasthenia gravis, cerebellar degeneration,
neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS),
and sensory neuropathy, multifocal motor neuropathy, Sheehan's
syndrome, autoimmune hepatitis, chronic hepatitis, lupoid
hepatitis, giant-cell hepatitis, chronic active hepatitis or
autoimmune chronic active hepatitis, pneumonitis such as lymphoid
interstitial pneumonitis (LIP), bronchiolitis obliterans
(non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease
(IgA nephropathy), idiopathic IgA nephropathy, linear IgA
dermatosis, acute febrile neutrophilic dermatosis, subcorneal
pustular dermatosis, transient acantholytic dermatosis, cirrhosis
such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune
enteropathy syndrome, Celiac or Coeliac disease, celiac sprue
(gluten enteropathy), refractory sprue, idiopathic sprue,
cryoglobulinemia such as mixed cryoglobulinemia, amylotrophic
lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery
disease, autoimmune ear disease such as autoimmune inner car
disease (AIED), autoimmune hearing loss, polychondritis such as
refractory or relapsed or relapsing polychondritis, pulmonary
alveolar proteinosis, keratitis such as Cogan's
syndrome/nonsyphilitic interstitial keratitis, Bell's palsy,
Sweet's disease/syndrome, rosacea autoimmune, zoster-associated
pain, amyloidosis, a non-cancerous lymphocytosis, a primary
lymphocytosis, which includes monoclonal B cell lymphocytosis
(e.g., benign monoclonal gammopathy and monoclonal gammopathy of
undetermined significance, MGUS), peripheral neuropathy,
paraneoplastic syndrome, channelopathies such as epilepsy,
migraine, arrhythmia, muscular disorders, deafness, blindness,
periodic paralysis, and channelopathies of the CNS, autism,
inflammatory myopathy, focal or segmental or focal segmental
glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis,
chorioretinitis, autoimmune hepatological disorder, fibromyalgia,
multiple endocrine failure, Schmidt's syndrome, adrenalitis,
gastric atrophy, presenile dementia, demyelinating diseases such as
autoimmune demyelinating diseases and chronic inflammatory
demyelinating polyneuropathy, Dressler's syndrome, alopecia areata,
alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon,
esophageal dysmotility, sclerodactyl), and telangiectasia), male
and female autoimmune infertility, e.g., due to anti-spermatozoan
antibodies, mixed connective tissue disease, Chagas' disease,
rheumatic fever, recurrent abortion, farmer's lung, erythema
multiforme, post-cardiotomy syndrome, Cushing's syndrome,
bird-fancier's lung, allergic granulomatous angiitis, benign
lymphocytic angiitis, Alport's syndrome, alveolitis such as
allergic alveolitis and fibrosing alveolitis, interstitial lung
disease, transfusion reaction, leprosy, malaria, parasitic diseases
such as leishmaniasis, trypanosomiasis, schistosomiasis,
ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome,
dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial
pulmonary fibrosis, interstitial lung fibrosis, fibrosing
mediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis,
cystic fibrosis, endophthalmitis, erythema elevatum et diutinum,
erythroblastosis fetalis, eosinophilic fasciitis, Shulman's
syndrome, Felty's syndrome, flariasis, cyclitis such as chronic
cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic),
or Fuch's cyclitis, Henoch-Schonlein purpura, human
immunodeficiency virus (HIV) infection, SCID, acquired immune
deficiency syndrome (AIDS), echovirus infection, sepsis (systemic
inflammatory response syndrome (SIRS)), endotoxemia, pancreatitis,
thyroxicosis, parvovirus infection, rubella virus infection,
post-vaccination syndromes, congenital rubella infection,
Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune
gonadal failure, Sydenham's chorea, post-streptococcal nephritis,
thromboangitis obiterans, thyrotoxicosis, tabes dorsalis,
chorioiditis, giant-cell polymyalgia, chronic hypersensitivity
pneumonitis, conjunctivitis, such as vernal catarrh,
keratoconjunctivitis sicca, and epidemic keratoconjunctivitis,
idiopathic nephritic syndrome, minimal change nephropathy, benign
familial and ischemia-reperfusion injury, transplant organ
reperfusion, retinal autoimmunity, joint inflammation, bronchitis,
chronic obstructive airway/pulmonary disease, silicosis, aphthae,
aphthous stomatitis, arteriosclerotic disorders (cerebral vascular
insufficiency) such as arteriosclerotic encephalopathy and
arteriosclerotic retinopathy, aspermatogenesis, autoimmune
homolysis, Boeck's disease, cryoglobulinemia, Dupuytren's
contracture, endophthalmia phacoanaphylactica, enteritis allergica,
erythema nodosum leprosum, idiopathic racial paralysis, chronic
fatigue syndrome, febris rheumatica, Hamman-Rich's disease,
sensoneural hearing loss, haemoglobinuria paroxysmatica,
hypogonadism, ileitis regionalis, leucopenia, mononucleosis
infectiosa, traverse myelitis, primary idiopathic myxedema,
nephrosis, ophthalmia symphatica (sympathetic ophthalmitis),
neonatal ophthalmitis, optic neuritis, orchitis granulomatosa,
pancreatitis, polyradiculitis acuta, pyoderma gangrenosum,
Quervain's thyreoiditis, acquired spenic atrophy, non-malignant
thymoma, lymphofollicular thymitis, vitiligo, toxic-shock syndrome,
food poisoning, conditions involving infiltration of T cells,
leukocyte-adhesion deficiency, immune responses associated with
acute and delayed hypersensitivity mediated by cytokines and
T-lymphocytes, diseases involving leukocyte diapedesis, multiple
organ injury syndrome, antigen-antibody complex-mediated diseases,
antiglomerular basement membrane disease, autoimmune
polyendocrinopathies, oophoritis, primary myxedema, autoimmune
atrophic gastritis, rheumatic diseases, mixed connective tissue
disease, nephrotic syndrome, insulitis, polyendocrine failure,
autoimmune polyglandular syndromes, including polyglandular
syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH),
cardiomyopathy such as dilated cardiomyopathy, epidermolisis
bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic
syndrome, primary sclerosing cholangitis, purulent or nonpurulent
sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary,
or sphenoid sinusitis, allergic sinusitis, an eosinophil-related
disorder such as eosinophilia, pulmonary infiltration eosinophilia,
eosinophilia-myalgia syndrome, Loffler's syndrome, chronic
eosinophilic pneumonia, tropical pulmonary eosinophilia,
bronchopneumonic aspergillosis, aspergilloma, or granulomas
containing eosinophils, anaphylaxis, spondyloarthropathies,
seronegative spondyloarthritides, polyendocrine autoimmune disease,
sclerosing cholangitis, sclera, episclera, chronic mucocutaneous
candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of
infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome,
angiectasis, autoimmune disorders associated with collagen disease,
rheumatism such as chronic arthrorheumatism, lymphadenitis,
reduction in blood pressure response, vascular dysfunction, tissue
injury, cardiovascular ischemia, hyperalgesia, renal ischemia,
cerebral ischemia, and disease accompanying vascularization,
allergic hypersensitivity disorders, glomerulonephritides,
reperfusion injury, ischemic re-perfusion disorder, reperfusion
injury of myocardial or other tissues, lymphomatous
tracheobronchitis, inflammatory dermatoses, dermatoses with acute
inflammatory components, multiple organ failure, bullous diseases,
renal cortical necrosis, acute purulent meningitis or other central
nervous system inflammatory disorders, ocular and orbital
inflammatory disorders, granulocyte transfusion-associated
syndromes, cytokine-induced toxicity, narcolepsy, acute serious
inflammation, chronic intractable inflammation, pyelitis,
endarterial hyperplasia, peptic ulcer, valvulitis, and
endometriosis.
[0201] B.5 Formulation
[0202] For use in the treatment of a disease, a 2H7 antibody
purified by the crystallization method of the present invention can
be prepared into a liquid formulation comprising the antibody at
about 20 mg/ml, 20 mM sodium acetate, 4% trehalose dihydrate, 0.02%
polysorbate 20, pH 5.5, for intravenous administration. A liquid
formulation comprising humanized 2H7 antibody at about 20 mg/ml, in
20 mM sodium acetate, 240 mM (8%) trehalose dihydrate, pH 5.3,
0.02% Polysorbate 20 is also provided. The 2H7 antibody can also be
formulated for subcutaneous administration in a formulation
comprising about 150 mg/ml antibody in 30 mM sodium acetate, pH
5.3, 7% trehalose dehydrate, 0.02% polysorbate 20 (Tween
20.RTM.).
[0203] Further details of the invention are provided in the
following non-limiting Examples.
[0204] All patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties.
EXAMPLES
[0205] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Commercially available reagents referred to
in the examples were used according to manufacturer's instructions
unless otherwise indicated. The source of those cells identified in
the following examples, and throughout the specification, by ATCC
accession numbers is the American Type Culture Collection,
Manassas, Va. In the examples below, 2H7 refers to humanized 2H7
antibody variant A, unless indicated otherwise.
Example 1
Dialysis Crystallization Studies
1. Effect of PBS Concentration on Dialysis Crystallization of
2H7
[0206] Materials and Methods for Dialysis Studies
[0207] 1. 150 mg/ml 2H7 drug substance
[0208] 2. Pierce Slide-Alyzer.RTM. Dialysis Cassette, 30 kDa
cutoff
[0209] 3, PBS 20.times. and 1.times.
[0210] 4. 1 L glass beaker
[0211] The glass beaker with stir bar was filled with 1 L PBS. Per
vendor instructions, cassette was presoaked for 30 sec with PBS,
then filled with 3 ml of 2H7 using an 181/2 gauge needle. The
cassette was floated in the beaker and the top was covered with
aluminum Coil. Upon end of experiment, the cassette was removed,
and any supernatant was removed with an 181/2 gauge syringe. The
cassette was then cut open along the edge of the membrane, and the
remaining material was scrapped off the membrane film using a
spatula.
[0212] 20.times. and 1.times.PBS were used to make 0.1.times.,
1.times., 10.times. and 20.times. solutions. 150 mg/ml bulk
antibody (2H7 drug substance) was dialyzed into beakers containing
each PBS concentration. All experiments were performed at
37.degree. C.
[0213] Composition of 2H7 drug substance (aka Formulated Bulk):
150 mg/ml 2H7
30 mM Sodium Acetate, pH 5.3
[0214] 7% Trehalose dehydrate
0.02% Polysorbate 20
[0215] Results
[0216] Table 2 shows the visual observations of each cassette after
20 hours. FIG. 1 shows precipitate from the 20.times. case.
Material placed on microscope slides for observation created a thin
film that dried and cracked while observing under the microscope
(FIG. 2).
TABLE-US-00018 TABLE 2 Effect of PBS Concentration on Dialysis
Crystallization of 2H7 bulk @ t = 20 h PBS Concentration
Observations 0.1X No change, no crystallization 1X 10% of the
cassette had small white precipitate. Cloudiness and long strips of
translucent precipitate as well 10X Bubbles, totally white and
opaque 20X Thicker than protein in mixture in 10X cassette. White
at the air interface, the rest was milky and translucent
2. Effect of Temperature on Dialysis Crystallization of 2H7
[0217] Based on the previous experiment, 1.times.PBS was chosen for
this study. Experiments were performed at 4.degree. C., 24.degree.
C. and 37.degree. C., using a 2-8.degree. C. cold room, room
temperature, and incubator environments, respectively. Just as in
the previous experiment, crystallization was performed using 150
mg/ml 2H7 bulk.
[0218] Results
[0219] Table 3 highlights the microscope observations at the end of
24 hours.
TABLE-US-00019 TABLE 3 Microscope Observations: Effect of
Temperature on Dialysis Crystallization Temperature % Out of
(.degree. C.) solution Observations 4 100 A few needle-like
crystals (much smaller than at 24.degree. C. and 37.degree. C.) in
precipitate 24 45 Liquid layer has tiny needles visible to naked
eye. Solid, white layer is amorphous solid with small crystals
mixed within. Translucent later as larger crystals in amorphous
precipitate. 36.7 45 Liquid has crystalline needles, as seen with
24.degree. C. condition in the liquid. Solid, white layer is
amorphous solid. Translucent layer has amorphous solid with
cube-like crystals.
[0220] Discussion
[0221] It was conjectured that at first, a large amount of protein
falls out of solution, which decreases the protein concentration of
the solution. At this lower protein concentration, crystals can
then form. This appears to be the case in the 24.degree. C.
condition, where bands of white precipitate were forming at the
same time as translucent layers with what appeared to be crystals.
However, under the microscope, it appeared that crystals mixed into
the thick opaque white precipitate, but they could not be
isolated.
Example 2
PBS Batch Studies
[0222] Following the dialysis studies described in Example 1,
crystallization of 2H7 by direct mixing, also known at the batch
method, was studied, using PBS as the precipitant. The experiments
were designed to observe the reactions in direct mixing of lower
concentrations of 2H7 with PBS at the three temperature points used
in the dialysis experiments.
[0223] Batch Crystallization
[0224] In all batch crystallization studies, a 2H7 CD20 antibody
solution was added to a 5-ml tube and allowed to equilibrate at the
desired temperature. Precipitant solution (at the same temperature)
was added to the tube, and the mixture was rotated continuously in
the Lab Quake Tube shaker. At the end of the experiment, (typically
18+ hours), samples were observed under a microscope. The tubes
were then centrifuged. The supernatant was sterile filtered and
analyzed for antibody concentration.
[0225] Materials and Methods
[0226] 1. 150 mg/ml 2H7 bulk
[0227] 2. 2H7 buffer without Trehalose/Tween
[0228] 3. PBS at 20.times. and 1.times.
[0229] 4. 5 ml Falcon tubes BD Falcon Polystrene tubes
[0230] 5. Pall Acrodisc 13 mm syringe Filters 0.2 .mu.m Supor
membrane Pall #4602
[0231] 6. 5 ml syringe
[0232] 7. Lab Quake Tube Shaker
[0233] 8. Microcentrifuge tubes
[0234] 9. Shimadzu UV/VIS Spectrophotometer
[0235] The 2H7 solution was added to the 5 ml tube and allowed to
equilibrate at the given temperature. An amount of PBS at the same
temperature was added to the 5 ml tube, and the mixture was rotated
continuously in the Lab Quake Tube shaker. At the end of the
experiment, samples were observed under the microscope. 1 ml
samples were transferred from the 5 ml tubes to microcentrifuge
tubes and centrifuged for 10 min at 1000 rpm. The supernatant was
then filtered using a syringe and 13 mm filter into another
microcentrifuge tube. This solution was then diluted accordingly
with formulation buffer for UV/VIS analysis.
1. Protein to Precipitant Ratio at Three Temperature Points
[0236] 150 mg/ml 2H7 was diluted to concentrations ranging from
5-100 mg/ml, s using 30 mM sodium acetate buffer. The ratio of 2H7
antibody solution to 1.times.PBS was varied, and the experiments
were performed at 4.degree. C., 24.degree. C., and 37.degree. C.
The results were observed over 24 hours.
[0237] Results
[0238] No changes were observed at any conditions. All tubes
remained clear.
[0239] Discussion
[0240] A higher concentration of PBS might be needed in the case of
direct mixing,
2. Protein to Precipitant Ratio at Three Temperature Points
[0241] The previous experiment was repeated using 10.times.PBS made
from diluting 20.times.PBS with DI water. Observations were carried
till day 4.
[0242] Results
[0243] Crystals were seen at all temperatures. They varied greatly
in size and shape, as noted in Table 4. A summary of Day 4
microscope observations grouped by final 2H7 concentration in the
solution is shown in Table 5.
TABLE-US-00020 TABLE 4 ##STR00001##
TABLE-US-00021 TABLE 5 ##STR00002##
[0244] The darkly shaded cells represent the observation of an
amorphous solid precipitate. Empty cells represent no change. %
values show crystallization efficiency for each condition that
resulted in the formation of crystals.
[0245] Large "haystack" crystals grown at 24.degree. C. are seen in
FIG. 2, FIG. 3 is an example of the irregular heterogeneous
crystals seen 37.degree. C. The small needles formed at 4.degree.
C. are seen in FIG. 4.
[0246] Discussion
[0247] Crystallization was most readily seen at 37.degree. C.
conditions when the final 2H7 concentration in the tube was <50
mg/ml. At 24.degree. C., large haystack crystals were observed at
6.65 mg/ml and below. These crystals were large, but the
crystallization efficiency was low. The results also show that the
ratio of PBS to 2H7 solution (v/v) is not very significant.
Therefore, for simplicity, it was decided to use a 1:1 ratio of PBS
to 2H7 solution (v/v) for future experiments. Temperature was fixed
at 37.degree. C. for future experiments, as 2H7 crystallized at
this condition at a wide range of concentrations. In addition, this
temperature allows for the most flexibility in process design.
Example 3
Salt Screening Studies
[0248] The purpose of the salt screening studies was to identify
other salts that could be used to induce 2H7 crystallization. The
starting point was to look at individual salts that compose the PBS
buffer. Following these, other salts with similar properties were
tested.
[0249] Materials and Methods
[0250] 1. Tris-HCl
[0251] 2. Tris-Base
[0252] 3. NaCl
[0253] 4. Na.sub.2SO.sub.4
[0254] 5. KCl
[0255] 6. K.sub.2SO.sub.4
[0256] 7. Na.sub.2HPO.sub.4
[0257] 8. KH.sub.2PO.sub.4
[0258] 9. 30 nM Sodium Acetate buffer
[0259] 10. 2H7 drug substance
[0260] 100 ml of 1M stock solutions were prepared in a 20 mM
Tris-HCl buffer for each salt. These stock solutions were diluted
to the desired concentrations for each experiment using 20 mM
Tris-HCL. The final salt concentrations were half of the starting
solutions as they were diluted 1:1 when combined with the 2H7
solutions. Five concentration dilutions of 2H7 bulk were made using
Sodium Acetate buffer. Crystallization experiments were run at
37.degree. C. using the same procedure as the batch studies
described in Example 2. 10.times.PBS was run as a positive control,
and 20 mM Tris-HCl buffer as a negative control,
1. Salt Screen
[0261] 500 mM and 1M made for NaCl, Na2SO4, KCL and
K.sub.2SO.sub.4
[0262] Results
[0263] Some crystallization was seen for KCl and NaCl.
Na.sub.2SO.sub.4 cases created a mixture of precipitate and
crystals. It appeared that there was crystallization after the
majority of the 2H7 fell out of solution as precipitate. This was
similar to what was observed in the 10.times. PBS dialysis
experiment. No change was seen with the K.sub.2SO.sub.4. The
crystal morphology of 10.times.PBS crystals varied greatly with the
protein concentration. Large round crystals mixed with needles were
seen at higher concentrations, while needles were formed at lower
concentrations (FIGS. 5 and 6).
2. Phosphate Salt Screen
[0264] 300 mM and 1M solutions were made for KH.sub.2PO.sub.4 and
Na.sub.2HPO.sub.4.
[0265] Results
[0266] Crystallization was observed using both salts. The
Na.sub.2HPO.sub.4 crystals were primarily thinneedles as shown in
FIG. 7. The length of the needles was inversely proportional to the
2H7 concentration. A new peanut shape was observed with the KH2PO4
salt (FIG. 8). Some of the peanuts were even clustered in larger
globular formations (FIG. 9). This shape is preferable over the
thin needles typically seen, because they are thicker, and
presumably more robust. The results of microscopic observations are
summarized in Table 6. Table 7 shows that the crystallization
efficiency of the Na.sub.2HPO.sub.4 cases is much lower than those
of KH.sub.2PO.sub.4 experiments.
TABLE-US-00022 TABLE 6 ##STR00003##
[0267] In the foregoing Table, dark shaded cells represent no
crystallization, and empty cells represent no change. Grey shade
represents a mixture of precipitate and crystals. AS=amorphous
solid.
TABLE-US-00023 TABLE 7 Salt Screen Studies - Summary of
Crystallization Efficiencies NaCl 1 M 37.5 7 KCl 1 M 75 84 KCl 1 M
15.5 52 KCl 500 mM 75 84 KH.sub.2PO.sub.4 300 mM 75 97
KH.sub.2PO.sub.4 300 mM 37.5 54 KH.sub.2PO.sub.4 300 mM 17.5 77
KH.sub.2PO.sub.4 1 M 75 98 KH.sub.2PO.sub.4 1 M 37.5 95
KH.sub.2PO.sub.4 1 M 17.5 92 Na.sub.2HPO.sub.4 300 mM 75 76
Na.sub.2HPO.sub.4 300 mM 37.5 54 Na.sub.2HPO.sub.4 300 mM 17.5 3
PBS 10X 75 94 PBS 10X 37.5 93 PBS 10X 17.5 88 PBS 10X 5 59
[0268] Discussion
[0269] Based on crystallization efficiency and range of possible
2H7 concentrations, the results suggest that the best
crystallization is seen with KH.sub.2PO.sub.4 and PBS. The 1M
KH.sub.2PO.sub.4 conditions yielded >90% crystallization between
75 and 17.5 mg/ml. These crystals had a peanut shape that appears
to be more robust than the needles seen with the control. The
control, 10.times.PBS produced largely thin, needle shaped
crystals, however crystallization was observed over the largest
range of protein concentrations; 75-5 mg/ml. The other individual
components of PBS; KCl, Na.sub.2HPO.sub.4 and NaCl did not show
similar crystallizing properties, which is unexpected. Upon further
investigation, it was noted that the largest component of PBS is
NaCl, which exhibited the lowest level of crystallizing properties.
Future experiments were designed to look at both PBS and
KH.sub.2PO.sub.4 conditions.
Example 4
Comparison of 2H7 with and without Trehalose and Polysorbate
[0270] The 2H7 used in the previous experiments was from the final
2H7 drug substance, which contains both polysorbate (TWEEN.RTM.) 20
and trehalose. It was thought that these components could have a
confounding, effect on crystallization. In order to investigate the
effects of these components, a pool of 2H7 antibody was
concentrated that had been run through the Q-Sepharose
chromatography step. This Q-Pool (Q-herein refers to Q-Sepharose)
was concentrated using ultrafiltration (UF). Unlike the final bulk
material, TWEEN.RTM. 20 and trehalose were left out of the
formulation. The Q-Step is typically the final chromatography step
and precedes concentration and formulation via
ultrafiltration/diafiltration (UF/DF). The concentrated Q-pool
material was then to be compared side-by-side with the bulk
product.
[0271] Materials and Methods
[0272] 1. 2H7Bulk material (2H7 drug substance)
[0273] 2. 2H7 Concentrated Q-Pool at 169.7 mg/ml
[0274] 3. Sodium acetate buffer
[0275] Both bulk and concentrated material were diluted with sodium
acetate buffer to yield the following starting concentrations when
combined 1:1 with precipitant:
TABLE-US-00024 D0 D1 D2 D3 D4 150 37.5 17.5 5 1.5
[0276] The method described in the batch studies (Example 1) was
used to crystallize the protein.
Composition of Q Sepharose pool: 169.7 mg/ml 2H7
20 mM Sodium Acetate, pH 5.3
1. 10.times.PBS Screen+/-TWEEN and Trehalose
[0277] 10.times.PBS used as precipitant to compare both types of
2H7 at 5 concentrations
Results
TABLE-US-00025 [0278] TABLE 8 Effects of TWEEN and trehalose, 1-X
PBS crystallization Starting Concentration (mg/ml) Trehalose/TWEEN
Crystallization efficiency 75 - 98% 75 + 94% 37.5 - 94% 37.5 + 94%
17.5 - 91% 17.5 + 90% 5 - 77% 5 + 61% 1.5 - 4% 1.5 + --
[0279] Table 8 highlights the crystallization efficiencies seen in
both the presence and absence of TWEEN and trehalose. FIGS. 10A-H
compare the crystals morphologies for each concentration.
[0280] Discussion
[0281] The presence of TWEEN.RTM. and trehalose seems to have a
negligible effect on crystallization efficiency. This was the first
time crystallization was observed at the 1.5 mg/ml concentration.
This was only in the case without trehalose and TWEEN.RTM.. Since
only 4% crystallized, and this was a single experiment, there is
not enough evidence to conclusively say that 2H7 without
TWEEN.RTM./trehalose crystallizes protein at lower concentrations.
The trehalose/TWEEN.RTM. does have a significant effect on size and
morphology of the crystals. As seen in FIGS. 10A-H, the bulk 2H7
formed bigger crystals at all protein concentrations. Most notably,
at 5 mg/ml, long 0.5 mm needles are formed with
TWEEN.RTM./trehalose, and 0.05 mm microneedles in the absence of
TWEEN.RTM./trehalose,
2. KH.sub.2PO.sub.4 Crystallization+/-TWEEN.RTM. and Trehalose
[0282] 1M KH.sub.2PO.sub.4 solution was used as precipitant to
compare both types of 2H7 at 5 concentrations. The results are
shown in FIGS. 11A-E. FIGS. 11A-E capture the effects of TWEEN.RTM.
and trehalose on crystal morphology. Table 9 below shows the
differences in crystallization efficiency. The presence of
TWEEN.RTM. and trehalose had negligible effects on crystallization
efficiency, but has a significant effect on size and morphology of
crystals. At 37.5 mg/ml, peanut and teardrop shaped crystals were
seen in the 2H7 bulk, while the absence of TWEEN.RTM. and trehalose
resulted in small, mealy, irregular crystals. Precipitation was
seen at 500 mM, KH.sub.2PO.sub.4--Trehalose 75 mg/l.
TABLE-US-00026 TABLE 9 Effects of TWEEN .RTM. and trehalose, 500 mM
KH.sub.2PO.sub.4 crystallization Starting Concentration (mg/ml)
Trehalose/TWEEN Crystallization efficiency 75 + 98% 75 - 99% 37.5 +
96% 37.5 - 98% 17.5 + 90% 17.5 - 94% 5 + 69% 5 - 88%
[0283] Summary
[0284] The data from this study is conclusive in finding that
TWEEN.RTM./trehalose do not affect crystallization efficiency (FIG.
12). The data also suggests that the TWEEN.RTM./trehalose affects
the crystal size and morphology. Bigger, more uniform crystals were
observed in the presence of TWEEN.RTM./trehalose in both KH2PO4 and
10.times.PBS cases. Most likely, the TWEEN.RTM. is the cause of
this difference. Trehalose is a sugar used for cryogenic protection
of the protein during freezing and thawing of bulk material.
POLYSORBATE.RTM. 20 is a nonionic surfactant added to the majority
of antibody drug formulations and serves to protect the proteins in
these drugs from denaturation and aggregation. Nonionic detergents
such as this contain a hydrophobic region which is derived from
fatty acid triglycerides. It is possible that the TWEEN.RTM. aids
in the formation of the crystal lattice which is driven by
hydrophobic interactions. It is possible that the morphology and
size of crystals can be manipulated by adding TWEEN.RTM. or other
detergents.
Example 5
Crystallization Out of Harvested Cell Culture Fluid (HCCF)
[0285] Up until this point, 2H7 hulk and concentrated Q-pool were
successfully crystallized. Both of these antibody sources are
highly purified. In order to pursue the goal of evaluating
feasibility of using crystallization to eliminate one or more
chromatography steps in the purification process, crystallization
of 2H7 from less purified material was examined. Ideally, 2H7 would
be crystallized directly from Harvested Cell Culture Fluid (HCCF).
This is the material that enters the purification process after the
cell culture process is ended and the cells are removed from the
fluid containing secreted 2H7 via centrifugation. If 2H7 could be
crystallized from HCCF, we could most likely crystallize the
protein at any step in the process. The 2H7 HCCF obtained had a
titer of 1.44 mg/ml at the time of harvest. Given that we had not
seen consistent crystallization of purified 2H7 at this
concentration, some of this material was concentrated using
ultrafiltration (UF). The smallest working volume for this
equipment was around 500 ml, and we had approximately 10 L of
material so we were limited to a maximum concentration of the HCCF.
For practical applications, concentration of HCCF much more than
10.times. would be undesirable due to the time of the operation.
Estimating from the Q-pool and bulk experiments, recovery >60%
can be expected if the antibody was concentrated to approximately
11.times.. For these reasons, 10 L of HCCF was concentrated to 15.5
mg/ml.
[0286] Materials and Methods
[0287] 1. 2H7 HCCF concentrated
[0288] 2. 2H7 HCCF
[0289] 3. KH.sub.2PO.sub.4
[0290] 4. PBS
[0291] Frozen HCCF was filtered through a 0.2 .mu.m filter after
thaw. Dilutions of HCCF were made using the concentrated HCCF and
straight HCCF from the same lot. The same batch study method of
crystallization was used in this study. At the end of
crystallization, protein concentration of the supernatant was
measured using Pro Sep A chromatography.
1. HCCF Crystallization Proof of Concept Run
[0292] 1 M and 1.5 M KH.sub.2PO.sub.4 solutions and 20.times.,
15.times., and 10.times.PBS solutions were used.
[0293] HCCF was at 15.5 mg/ml in all cases.
[0294] 15.5 mg/ml Q-Pool was used as a control.
[0295] Results
[0296] Crystallization was observed at 20.times., 15.times., and
10.times.PBS and KH.sub.2PO.sub.4 at both concentrations. The
higher PBS concentrations come from stock solutions of PBS buffer
that have not been diluted to the typical 1.times. working
concentration. There were noticeable differences in crystal
morphology and size (FIGS. 13A-H).
2. HCCF Crystallization Screen
[0297] HCCF dilutions made between 15.5 and 1.44 mg/ml
[0298] 2M-200 mM KH.sub.2PO.sub.4 solutions
[0299] 20.times.-1.times.PBS
[0300] Results
[0301] Crystallization was observed at PBS concentrations ranging
from 5.times. to 20.times., and KH2PO4 concentrations between 1.5M
and 1M (Table 10).
TABLE-US-00027 TABLE 10 HCCF Crystallization Screen Precipitant 2H7
starting Crystallization Precipitant concentration concentration
efficiency KH.sub.2PO.sub.4 500 mM 2.5 58% KH.sub.2PO.sub.4 500 mM
4.25 74% KH.sub.2PO.sub.4 500 mM 7.75 98% KH.sub.2PO.sub.4 750 mM
1.5 54% KH.sub.2PO.sub.4 750 mM 2.5 <10% PBS 5X 2.5 5% PBS 5X
4.25 70% PBS 10X 4.25 71% PBS 10X 7.75 90% PBS 15X 2.5 70% PBS 15X
4.25 80% PBS 15X 7.75 91% PBS 20X 2.5 77% PBS 20X 4.25 90% PBS 20X
7.75 87%
3. HCCF pH Screen
[0302] HCCF dilutions made between 15.5 and 1.44 mg/ml
[0303] 10.times.PBS solutions prepared at pH, 6, 6.5, 7, 7.7 and
8.
[0304] 500 mM KH.sub.2PO.sub.4 prepared at pH 6, 6.5, 7, 7.7 and
8.
[0305] Results
[0306] 2H7 will crystallize to varying degrees over pH's ranging
from 6.0 and 8.0. The largest range concentrations crystallized
with 10.times.PBS was at pH 7 (Table 11). At pH 7, 0.77 mg/ml,
4.25, and 7.75 mg/ml crystallized, but 1.5 mg/ml did not. This was
the first time crystallization was observed at 0.77 mg/ml, which
was the non-concentrated HCCF fluid. Crystallization efficiency at
that concentration was low, 29%, and this outcome was not
repeatable. The low yields and range of crystallization for
10.times.PBS at pH 6.5 is unusual given that the unadjusted pH is
6.7. Crystallization was achieved over a wide range of pH values
and protein concentrations with 500 mM KH.sub.2PO.sub.4. At 7.5 and
8, 2H7 crystallized at the largest range of concentrations, between
1.5 and 7.75 mg/ml (see Table 12). Table 13 highlights the crystal
morphologies observed at different concentrations of PBS and
KH.sub.2PO.sub.4.
TABLE-US-00028 TABLE 11 HCCF pH Screen - 10X PBS pH Initial 2H7
Conc. (mg/ml) Crystallization Efficiency 6 4.25 57% 6 7.75 77% 6.5
5.25 67% 6.5 7.75 84% 7 0.77 29% 7 4.25 70% 7 7.75 84% 7.5 4.25 74%
7.5 4.25 74% 8 4.25 73% 8 7.75 82%
TABLE-US-00029 TABLE 12 HCCF pH Screen - 500 mM KH.sub.2PO.sub.4 pH
Initial 2H7 Crystallization Efficiency 6 4.25 50% 6 7.75 88% 6.5
4.25 81% 6.5 7.75 84% 7 2.5 88% 7 4.25 92% 7 7.75 92% 7.5 1.5 83%
7.5 2.5 88% 7.5 4.25 93% 7.5 7.75 94% 8 1.5 20% 8 2.5 89% 8 4.25
77% 8 7.75 73%
TABLE-US-00030 TABLE 13 Effects of pH on HCCF Crystal Morphology pH
500 mM KH2PO4 10X PBS 6.0 Only thin needles at 15.5 mg/ml Long
needles + 100 .mu.m at 8.5 mg/ml short needles at 15.5 mg/ml 6.5
Long needles at 8.5 and 15.5 mg/ml Long needles at 8.5 mg/ml, short
needles at 15.5 mg/ml 6.7 (Std PBS) Needles and needle fragments
(20-100 .mu.m) at 3-15.5 mg/ml 7.0 Needles at 5, 8.5, and 15.5
mg/ml Long needles at 8.5 mg/ml, Micro-needles at 15.5 mg/ml 7.2
(Std, KH.sub.2PO.sub.4) Thick needles and haystacks (20-50 .mu.m)
at 5, 8.5 and 15.5 mg/ml 7.5 Short needles at 3 mg/ml, Needles at
8.5 mg/ml, irregular clusters at 5, 8.5 micro-needles at 15.5 mg/ml
and 15.5 mg/ml 8.0 Short needles at 1.44 mg/ml, Needles at 8.5
mg/ml, irregular clusters, balls and micro-needles at 15.5 mg/ml
peanuts at 3-15.5 mg/ml
[0307] Discussion
[0308] Comparing overall crystallization efficiencies of the two
salts, KH.sub.2PO.sub.4 has consistently higher yields at each pH
value. The highest values were seen at 7.5, where all
concentrations had yields >80% and 2 cases >92%. In
comparison, none of the 10.times.PBS cases had a yield greater than
84%. Crystallization efficiency increases with protein
concentration. With an increase in pH, 500 mM KH.sub.2PO.sub.4 was
effective in crystallizing 2H7 at lower concentrations. At pH 6,
crystallization was only seen at 4.25 mg/ml and above. At pH 7.5,
HCCF crystallized at all concentrations tested with the exception
of 1.times.. It is also interesting to note that there seems to be
a peak of effectiveness somewhere between pH 7.5 and 8.0, as noted
with a decrease in yields.
[0309] The pH also had an effect on the morphology of the crystals.
With PBS, needles were seen at all 4.25 and 7.75 mg/ml, however,
the needle length was consistently greater at the lower
concentration. This suggests that at a lower concentration there is
less nucleation and instead more growth and lengthening of existing
crystals. Many small crystals are characteristic of a rapid,
uncontrolled crystallization process. Looking at KH.sub.2PO.sub.4
conditions, as the pH increased, the morphology of the crystals
went from needles to irregular clusters and balls.
SUMMARY
[0310] In this HCCF crystallization study, it was found that
concentrated 2H7 readily crystallizes out of HCCF using the same
precipitants as identified to crystallize 2H7 bulk and concentrated
Q-Pool material. It was not possible to consistently crystallize
2H7 out of un-concentrated HCCF. As with bulk and concentrated
Q-pool material, lower crystallization efficiency was seen as the
2H7 concentration decreased. The pH of the precipitant has a
significant effect on the crystallization efficiency and
concentrations of 2H7 that will crystallize. 500 mM
KH.sub.2PO.sub.4 at pH 7.5 showed the highest yields of crystals
over the widest range of 2H7 concentrations.
[0311] It was also determined that the morphology and size of the
2H7 crystals from HCCF were better than those obtained from
concentrated Q-Pool and most comparable to crystals seen with 2H7
bulk. It is possible that the PLURONIC 68 detergent in the HCCF
media has an effect similar to the POLYSORBATE 20 found in the bulk
material. The morphology of the crystals also varied across the
range of precipitant pH values that were explored. Thus, pH is a
parameter that can be manipulated to achieve a morphology best
suited for downstream processing.
[0312] From this study forward, we will use KH.sub.2PO.sub.4
exclusively. From a scale-up perspective, the material requirements
for PBS are much greater than that of KH.sub.2PO.sub.4. PBS also
contains NaCl at high concentrations which can react with the
stainless steel tanks used in manufacturing. We also observed
comparable results in the HCCF crystallization proof of concept
screen and HCCF crystallization screen. In the HCCF pH screen, we
had the highest efficiencies, and greatest range of pH flexibility
and morphology with KH.sub.2PO.sub.4. In the following studies, we
look at further optimizing the precipitant conditions by looking at
tighter pH ranges and developing phase maps of the crystallization
process.
Example 6
Further Analysis of Process Parameters for Harvested Cell Culture
Fluid (HCCF) Process
[0313] After determining that 2H7 would readily crystallize from
HCCF, the focus has been shifted from bulk and Q-pool material to
crystallization from concentrated HCCF. From a purification
standpoint, it would be most useful to replace some of the
expensive and time and labor intensive upstream processes, like the
Protein A purification step (Pro-A), with crystallization. The goal
of this study is to further refine the precipitant conditions.
[0314] Materials and Methods
KH.sub.2PO.sub.4
Concentrated 2H7 HCCF
[0315] 2H7 HCCF from pH Optimization 500 mM KH.sub.2PO.sub.4
solutions at 6 points between 7 and 8 1.44 mg-8.5 mg/ml 2H7
concentrations Run in duplicate
Results
[0316] Crystallization was observed over a wide range of
concentrations. The highest crystallization efficiencies were seen
at the 8.5 mg/ml HCCF 2H7 concentration.
TABLE-US-00031 TABLE 14 HCCF pH Optimization Starting 3H7
Concentration Crystallization (mg/ml) KH.sub.2PO.sub.4 pH
efficiency Standard deviation 1.5 7.4 66% 0.23 1.5 7.6 73% 0.06 1.5
7.8 78% 0.05 1.5 8 81% 0.01 2.5 7 71% 0.22 2.5 7.2 69% 0.05 2.5 7.4
81% 0.08 2.5 7.6 84% 0.03 2.5 7.8 87% 0.04 2.5 8 86% 0.01 4.25 8
92% 0.00 4.25 7.2 81% 0.02 4.25 7.4 85% 0.03 4.25 7.6 90% 0.03 4.25
7.8 90% 0.02 4.25 8 91% 0.00 7.75 7 94% 7.75 7.2 88% 0.01 7.75 7.4
91% 0.01 7.75 7.6 92% 0.01 7.75 7.8 94% 0.01 7.75 8 95% 0.00
[0317] A graphic illustration of the HCCF crystallization
efficiency as a function of pH, using 500 mM KH.sub.2PO.sub.4, is
shown in FIG. 14.
[0318] Discussion
[0319] As seen in FIG. 14, at 2.5 mg/ml 2H7 and above, there is
little difference in crystallization efficiency between pH 7.6 and
8.0. The maximum and decline of crystallization efficiency between
pH 7.5 and 8 was not observed in this experiment. 2H7 at a
concentration of 1.5 mg/ml, did not crystallize at 7.0, and 7.2.
There was also a significant increase in crystallization efficiency
as the pH increased to 8.0. The final pH in each tube once the
KH.sub.2PO.sub.4 and HCCF were combined was consistently between
0.2-0.3 less than that of the KH.sub.2PO.sub.4 solution added.
[0320] Summary
[0321] Based on these experiments, for crystallization from CCF,
the optimal pH of K2PO4 is 7.8+/-0.2, and the optimal concentration
of the salt is 1 M. However, as the data show, other pHs and
concentrations also work.
Example 7
Dissolubility Studies
[0322] From the studies described in the previous Examples, the
regions have been determined in which precipitation and nucleation
of crystals are observed. Here, we will determine the metastable
region, where one no longer gets new crystal formation, but instead
sees crystal growth. To this end, we will determine the conditions
where crystals begin to dissolve back into solution. The
dissolubility studies aim at determining factors of
KH.sub.2PO.sub.4 pH and concentrations and developing solubility
curves that will complete the crystallization phase maps for
2H7.
[0323] Materials and Methods
1. 2H7 concentrated HCCF
2. 2H7 HCCF
3, KH.sub.2PO.sub.4
[0324] Crystals were prepared using the large batch method. The
supernatant from the tubes was removed using a benchtop aspirator.
Approximately 50 ml of KH.sub.2PO.sub.4 at pH 7.2 was added to the
falcon tube which was then shaken to resuspend the crystals in the
salt solution. This mixture was then centrifuged again and this
supernatant was exchanged for fresh KH.sub.2PO.sub.4. This process
was repeated 2.times..
[0325] After the third resuspension in KH.sub.2PO.sub.4, 2 ml of
this mixture was placed into 5 ml falcon tubes. The tubes were
centrifuged once, and the supernatant was aspirated and replaced
with 2 ml of KH.sub.2PO.sub.4 at the desired condition.
[0326] These tubes were placed in the rotator, and left 18+ hours.
At the end of this time, approximately 1 ml of the mixture was
filtered through a syringe with 13 mm filter into a microcentrifuge
tube. The protein concentration in solution was measured using Pro
A analysis.
1. KH.sub.2PO.sub.4 Concentration Dissolubility Study
[0327] KH.sub.2PO.sub.4 solutions at 7.8 ranging from 0.150M to
1.5M Water used as a control condition Concentrations measured at 1
h and 18 h 4.degree. C., Ambient temperature 24.degree. C., and
37.degree. C.
[0328] Results
[0329] The dissolubility curves are shown in FIG. 15-17.
[0330] Discussion
[0331] It was found that crystals redissolved in solutions at 750
mM and below. It appears that this takes place at the fastest rate
when incubated at 4.degree. C. with 26.7% dissolving within the
1.sup.st hour. This is consistent with our understanding that
crystallization is optimal at higher temperature. There seems to be
little difference between rates and percentages of dissolution for
24.degree. C. and 37.degree. C.
Example 8
Purification Starting from Concentrated HCCF with Crystallization
Step
[0332] Based on the previous experiments, the basic steps of the
crystallization unit operation, i.e. concentration,
crystallization, washing and dissolution, can replace two
chromatography steps. In this example, the starting material is
concentrated HCCF, which is run through this new 2H7 purification
process. Product purity and quality data are then collected and
compared with the traditional purification process.
[0333] Materials and Methods
1. Concentrated 2H7 HCCF
2. 1M K2HPO4
[0334] 3. 250 ml flask 4. Q-Sepharose buffer 5. Q-Sepharose
column
6. Centriprep
[0335] 70 ml of 2H7 HCCF at 15 g mg/ml crystallized in 250 ml flask
with 750 ml K2PO4 using large batch method. Crystals were washed
and dissolved into Q-Pool buffer using the optimized processes.
Samples were taken. The 2H7 pool purified using a Q-Sepharose
column at the given process conditions and samples were taken. The
Q-pool was concentrated using Centri-prep and samples were taken.
Samples were analyzed for titer, CHOP levels and aggregates.
[0336] Results
[0337] The centriprep was used to concentrate the antibody because
we were concentrating a small volume of material, <1 L. For a
volume greater than 1 L, a benchtop TFF can be used. Since the
UF/DF generally has little effect on the purity and quality of the
final product, the centriprep was seen as an acceptable
substitute.
TABLE-US-00032 TABLE 15 Purification Process Comparison -
Conventional vs. Crystallization Purity Levels CHO SEC 2H7/12K CHOP
LpA DNA (% Agg) Gentamycin Insulin feedstock ng/mg ng/mg ng/mg
(HMWS) ng/mg ng/mg HCCF 340,000-540,000 N/A 2500-4000 N/A
16,000-26,000 0.1 to 30 Prosep vA 1900-3100 8-13 1-2 1.1-1.3 7-13
LTD SP 550-650 <2 0.001-0.02 0.8-1.1 LTD LTD SEPHAROSE FF Q 6-10
<2 LTD 0.6-0.7 LTD LTD SEPHAROSE FF UFDF 3-6 <2 LTD 0.7-0.8
LTD to 0.03 LTD HCCF 340,000-540,000 N/A 2500-4000 N/A
16,000-26,000 0.1 to 30 Concentrated 3.4-5.4 8013 10-20 ?
160,000-260,000 1 to 300 HCCF million Crystallization 150-250 N/A
LTD 2.1-2.2 LTD LTD Q 6-10 <2 LTD 0.6-0.7 LTD LTD SEPHAROSE FF
Centriprep 3-6 <2 LTD 0.7-0.8 LTD LTD % Fragment (LMWS):
0.2-0.3%
[0338] Summary
[0339] The process was successful in purifying 2H7. The CHOP levels
were within the range of the standard process. The aggregate levels
were higher than in the current process, however, they were within
range of the Certificate Analysis for 2H7. This is, at least in
part, due to removing the SP-SEPHAROSE.RTM. step, which serves to
remove aggregates. It may be possible to optimize the
Q-SEPHAROSE.RTM. step to remove aggregates. Higher aggregates may
also be due to the shear rates for concentrating 2H7 HCCF. UF will
be investigated for any effect on aggregates.
Example 9
Effect of Potassium Phosphate Concentration on Crystallization of
Hu 2H7 Variant H
[0340] Tests were conducted with purified 2H7 variant H to see if
it would crystallize in similar conditions to 2H7 variant A (see
Table 1).
[0341] Materials and Methods
[0342] 1. 2H7 variant H unconditioned Bulk at 23 mg/ml (Material
that has been concentrated and diafiltered but has not had
trehalose or Tween.TM. added)
[0343] 2. 1 M Potassium Phosphate, pH 7.8
[0344] 3. Purified Water
[0345] The water and the potassium phosphate were mixed to make a
series of phosphate concentrations from 0-1.0 M. These along with
the unconditioned bulk were heated to 37.degree. C. then mixed 1:1,
and incubated at 37.degree. C. with mixing for 24 hours. Samples
were then centrifuged and the supernatants assayed for remaining
2H7 variant H concentration.
[0346] Results
TABLE-US-00033 TABLE 16 Effects of Potassium Phosphate
Concentration on Crystallization of 2H7 variant H Potassium
Phosphate Concentration (mM) Crystallization efficiency 0 0% 50
32.7% 100 71.2% 150 93.4% 200 96.6% 250 98.2% 300 99.0% 350 99.2%
400 99.6% 450 99.2% 500 99.8%
[0347] Discussion
[0348] This experiment confirmed that the conditions discovered for
2H7 variant A are applicable to 2H7 variant H. Although 2H7 variant
H showed similar crystallization behavior to variant A, it achieved
a near 100% crystallization efficiency at only 250 mM potassium
phosphate at pH 7.8.
Example 10
Effect of Potassium Phosphate Concentration on Crystallization of
Variant C
[0349] Tests were conducted with purified Variant C to see if it
would crystallize in similar conditions to 2H7 variant A.
[0350] Materials and Methods
[0351] 1. Variant C unconditioned Bulk at 25.3 mg/ml (material that
has been concentrated and diafiltered but has not had trehalose or
Tween added)
[0352] 2. 1 M Potassium Phosphate, pH 7.8
[0353] 3. Purified Water
[0354] The water and the potassium phosphate were mixed to make a
series of phosphate concentrations from 0-1.0 M. These along with
the unconditioned bulk were heated to 37.degree. C. then mixed 1:1,
and incubated at 37.degree. C. with mixing for 24 hours. Samples
were then centrifuged and the supernatants assayed for remaining
variant C concentration.
[0355] Results
TABLE-US-00034 TABLE 17 Effects of Potassium Phosphate
Concentration on Crystallization of variant C Potassium Phosphate
Concentration (mM) Crystallization efficiency 0 0% 50 26.9% 100
31.9% 150 83.8% 200 91.6% 250 95.8% 300 97.3% 350 98.3% 375 98.8%
400 99.1% 450 99.6% 500 99.7%
[0356] Discussion
[0357] This experiment confirmed that the conditions discovered for
humanized 2H7 variant A are applicable to variant C. Although
variant C showed similar crystallization behavior to variant A, it
achieved a near 100% crystallization efficiency at only 300 mM
potassium phosphate at pH 7.8.
Example 11
Effect of Potassium Phosphate Concentration and pH on
Crystallization of Variant C from Concentrated HCCF
[0358] Tests were conducted with variant C concentrated HCCF to
determine the effect of pH and phosphate concentration on variant C
crystallization and the resulting purification.
Materials and Methods
[0359] 1. variant C concentrated HCCF at 13.8 mg/ml,
1.8.times.10.sup.6 ng/mg host cell protein
[0360] 2. 1 M Potassium Phosphate, pH3, pH 4, pH5, pH 6, pH7, pH
8
[0361] 3. Purified Water
[0362] The water and the potassium phosphate were added to the
concentrated HCCF at 37.degree. C. to make a series of
crystallization experiments at phosphate concentrations between 0.2
and 0.5 M. These were done in 2 groups, the variant C concentration
was kept consistent in each, and hence the highest phosphate
concentration in each group determined final dilution for that
group. After mixing and incubation for greater than 24 hours, the
samples were centrifuged and the supernatants were measured for
residual variant C. For the samples from the first group, the
crystals were dissolved and the variant C and host cell protein
concentrations were measured to assess the purity after
crystallization.
TABLE-US-00035 TABLE 18 Effect of Potassium Phosphate Concentration
and pH on Crystallization of variant C from Concentrated HCCF
variant C starting Potassium concentration Crystallization HCP pH
Phosphate (mg/ml) efficiency ng/mg 8 300 mM 8.35 84.0% 133.0 8 363
mM 8.35 92.0% 292.0 8 417 mM 8.35 94.5% 322.0 7 300 mM 8.35 73.5%
196.0 7 363 mM 8.35 84.6% 275.0 7 417 mM 8.35 91.3% 210.0 6 300 mM
8.35 0.0% n/a 6 363 mM 8.35 48.2% 63.2 6 417 mM 8.35 68.3% 121.0 5
200 6.9 3.6% n/a 5 300 6.9 1.7% n/a 5 400 6.9 8.3% n/a 5 500 6.9
6.7% n/a 4 200 6.9 0.1%* n/a 4 300 6.9 27.6%* n/a 4 400 6.9 25.2%*
n/a 4 500 6.9 24.3%* n/a 3 200 6.9 97.3%* n/a 3 300 6.9 100%* n/a 3
400 6.9 100%* n/a 3 500 6.9 100%* n/a *precipitation
Discussion
[0363] Like 2H7 variant A, the concentration of phosphate required
to induce crystallization is reduced with increasing pH. At pH 5,
very little crystallization was observed at the phosphate
concentrations used. At pH 3 and 4, variant C precipitated rather
than crystallized. Precipitation differs from crystallization in
that it happens instantly upon mixing, the solid produced does not
settle, and cannot be redissolved. The level of host cell protein
was reduced to less than 0.2% of the starting material in all the
cases measured indicating that crystallization is an effective
purification tool.
Example 12
Application of Crystallization to the Purification of Variant C
from HCCF
[0364] Since variant C did not crystallize at pH 5 at potassium
phosphate concentrations that induced crystallization at higher pH,
diafiltration with 0.4 M potassium phosphate pH 5.0 was
incorporated into the initial HCCF concentration step.
Crystallization was then induced by adjusting concentrated HCCF to
pH 7.8.
Materials and Methods
[0365] 1. variant C HCCF at 1.8 mg/ml
[0366] 2. 0.4 M Potassium Phosphate, pH5
[0367] 3. Millipore Ultrafiltration Unit
[0368] A 10 L aliquot of variant C HCCF were concentrated .about.10
fold by ultrafiltration. It was then diafiltered with 5 diavolumes
of 0.4 M Potassium Phosphate pH 5.0. The concentrated variant C
HCCF was recovered from the system, adjusted to 37.degree. C. and
subsequently to pH 7.8. The sample was incubated at 37.degree. C.
with gentle mixing for 46 hours at which time the crystals were
recovered by centrifugation. Each batch of crystals was washed
twice with 0.4 M Potassium phosphate pH 8, then dissolved in 25 mm
Tris pH 8. The crystal pool had to be adjusted to pH 5.5 to achieve
complete dissolution.
Results
TABLE-US-00036 [0369] TABLE 19 Purification Results Step Yield (%)
Host Cell Protein (ng/mg) HCCF 100.0 87741.6 Concentrated HCCF
103.5 176856.6 Supernatant 7.7 748543.3 Wash 1 2.0 90226.9 Wash 2
0.7 42683.2 Dissolved Crystals 76.1 904.7
Discussion
[0370] The above crystallization procedure removed 99% of the host
cell proteins from the starting variant C HCCF. The yield of 76% is
comparable to the standard antibody process. Crystallizing the
antibody by diafiltration into the crystallization solution rather
than by direct addition of the crystallization solution to the
antibody solution allows maintenance of higher antibody
concentrations during crystallization. Diafiltration accomplishes
two functions--it is a method of exchanging into a different
buffer, in this case, from HCCF into the crystallization buffer
comprising the desired salt and pH, while at the same time
concentrating the HCCF solution. Because the concentration of
soluble antibody at the end of crystallization is independent of
the starting concentration, starting with higher antibody
concentrations increases the potential yield. Exchanging into the
crystallization buffer by diafiltration is especially useful when
the required concentration of crystallizing agent is near the
solubility limit of the agent. For example, it would be impossible
to conduct crystallization at the solubility of potassium phosphate
by diluting the antibody solution with concentrated potassium
phosphate, but this is achievable via diafiltration.
[0371] Conclusions
[0372] We have demonstrated that 2H7, a humanized monoclonal
antibody, and its variants can be crystallized. It has been
determined that the crystallization is optimal at increased
temperatures (4-40 C), in the presence of KH.sub.2PO.sub.4. 2H7 has
been crystallized from concentrated, purified bulk, concentrated
Q-Pool and concentrated HCCF. By optimizing the process conditions,
crystallization efficiencies of over 90% could be achieved from
concentrated HCCF. Because of the high level of purity, HCCF
crystallization can replace two of the longest, most expensive
chromatography steps, Protein A chromatography and SP-SEPHAROSE
chromatography. This was directly proven by purifying 1 gram of
2H7. The final product was comparable to the product seen with the
traditional process. Thus, crystallization is a feasible process
step for purifying 2H7 and its variants.
[0373] While the experiments were conducted with specific CD20
antibodies, the humanized 2H7 antibody variants, this approach is
equally suitable for crystallizing other CD20 antibodies,
including, without limitation, Rituximab (RITUXAN.RTM.), and the
2H7 variants specifically disclosed herein.
[0374] The invention illustratively described herein can suitably
be practiced in the absence of any element or elements, limitation
or limitations that is not specifically disclosed herein. Thus, for
example, the terms "comprising," "including," "containing," etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalent of
the invention shown or portion thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modifications and variations of
the inventions embodied herein disclosed can be readily made by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of the inventions
disclosed herein. The inventions have been described broadly and
generically herein. Each of the narrower species and subgeneric
groupings falling within the generic disclosure also form the part
of these inventions. This includes within the generic description
of each of the inventions a proviso or negative limitation that
will allow removing any subject matter from the genus, regardless
or whether or not the material to be removed was specifically
recited. In addition, where features or aspects of an invention are
described in terms of the Markush group, those schooled in the art
will recognize that the invention is also thereby described in
terms of any individual member or subgroup of members of the
Markush group. Further, when a reference to an aspect of the
invention lists a range of individual members, as for example, "SEQ
ID NO:1 to SEQ ID NO:100, inclusive," it is intended to be
equivalent to listing every member of the list individually, and
additionally it should be understood that every individual member
may be excluded or included in the claim individually.
[0375] From the description of the invention herein, it is manifest
that various equivalents can be used to implement the concepts of
the present invention without departing from its scope. Moreover,
while the invention has been described with specific reference to
certain embodiments, a person of ordinary skill in the art would
recognize that changes can be made in form and detail without
departing from the spirit and the scope of the invention. The
described embodiments are considered in all respects as
illustrative and not restrictive. It should also be understood that
the invention is not limited to the particular embodiments
described herein, but is capable of many equivalents,
rearrangements, modifications, and substitutions without departing
from the scope of the invention. Thus, additional embodiments are
within the scope of the invention and within the following claims.
All U.S. patents and applications; foreign patents and
applications; scientific articles; books; and publications
mentioned herein are hereby incorporated by reference in their
entirety as if each individual patent or publication was
specifically and individually indicated to be incorporated by
reference, including any drawings, figures and tables, as though
set forth in full.
Sequence CWU 1
1
151107PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 1Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser Asn Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr 85 90 95Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 1052122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr
Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Val Asp
Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val Tyr Tyr
Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 1203107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser
Val Ser Tyr Leu 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Trp Ala Phe Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 1054122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 4Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr
Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Val Asp
Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val Tyr Tyr
Ser Ala Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 1205122PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 5Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr
Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Val Asp
Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val Tyr Tyr
Ser Tyr Arg Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 1206213PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 6Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser
Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Phe Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
2107452PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 7Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe
Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345
350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
Lys 4508452PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 8Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly Lys 4509213PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser
Val Ser Tyr Leu 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser Asn Leu Ala Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Trp Ala Phe Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
21010452PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 10Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala
Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Ala Ser Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
45011452PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 11Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala
Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Ala Ser Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly Lys 45012452PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr
Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Val Asp
Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val Tyr Tyr
Ser Ala Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ala Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Ala Ala Leu Pro Ala Pro 325 330 335Ile Ala Ala Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
45013452PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 13Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala
Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Ala Ser Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Trp His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly Lys 45014452PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr
Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Val Asp
Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val Tyr Tyr
Ser Tyr Arg Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ala Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Ala Ala Leu Pro Ala Pro 325 330 335Ile Ala Ala Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
45015452PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 15Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala
Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys
Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly Lys 450
* * * * *